Neisseria meningitidis immunogenic compositions

ABSTRACT

Immunogenic compositions are disclosed that include  Neisseria meningitidis  microvesicles, such as outer membrane vesicles (OMV) and/or blebs, from PorA − PorB −  Neisseria, such as PorA − PorB − ampM −    Neisseria meningitidis.  These immunogenic compositions are of use to induce an immune response to  Neisseria,  including  Neisseria meningitidis  and  Neisseria gonorrhea.

CROSS REFERENCE TO RELATED APPLICATION(S)

This is a continuation of U.S. patent application Ser. No. 16/632,278,filed on Jan. 17, 2020, which is a § 371 U.S. national stage ofInternational Application No. PCT/US2018/043054, filed Jul. 20, 2018,which claims the benefit of U.S. Provisional Application No. 62/535,627,filed Jul. 21, 2017, which is incorporated herein by reference.

FIELD OF THE DISCLOSURE

This relates to immunogenic compositions comprising outer membranemicrovesicles, which include outer membrane vesicles (OMVs) and/orblebs, from PorA⁻PorB⁻ Neisseria, such as PorA⁻PorB⁻RmpM⁻ Neisseria (N.)meningitidis, that are of use to induce an immune response to Neisseria,including N. meningitidis and N. gonorrhoeae.

BACKGROUND

Neisseria is a genus of Gram-negative bacteria that colonize the mucosalsurfaces of many animals. There are eleven species that colonize human,of which two, N. meningitidis and N. gonorrhoeae, are pathogenic. N.meningitidis is the causative agent of meningitis and meningococcalsepticemia. N. gonorrhoeae is the causative agent of gonorrhea. Thegenomes of at least ten of the Neisseria species have been completelysequenced.

Diseases caused by N. meningitidis and N. gonorrhoeae are significanthealth problems worldwide; control of these diseases by developingmeningococcal and gonococcal vaccines is a public health priority.However, development of vaccines has been challenging.

N. meningitidis remains a significant cause of global morbidity andmortality, despite the availability of serogroup A-, C-, W-, andY-specific capsular polysaccharide (Ps) vaccines. Disease can be causedby meningococcal serogroup B (MenB) strains, which in the United Statesaccount for one-third of all invasive N. meningitidis infections and 60%of those in infants (Prevention, C.f.D.C.a., (2015) MeningococcalDisease. In: Epidemiology and Prevention of Vaccine-PreventableDiseases. J. Hamborsky, A. Kroger & C. Wolfe (eds). Washington, D.C.:Public Health Foundation, pp. 231-246). Unlike the other meningococcalserogroups, the capsule of MenB is poorly immunogenic (Wyle et al.(1972). J Infect Dis 126: 514-521), a result of its resemblance to apolysialylated Ps moiety present on human neural cells (Finne et al.(1987) J Immunol 138: 4402-4407). Efforts for vaccine design havefocused on identification of subcapsular antigens, includingsurface-expressed outer membrane proteins (OMPs). However, a needremains for additional vaccines. In addition, N. gonorrhoeae is anunencapsulated bacterium and currently no effective gonococcal vaccinesexist. A need remains for additional compositions that can be used toinduce an immune response to N. meningitidis and N. gonorrhoeae.

SUMMARY OF THE DISCLOSURE

Disclosed are isolated PorA⁻PorB⁻RmpM⁻ Neisseria (N.) meningitidis (alsocalled ΔPorAΔPorBΔRmpM) and compositions including an effective amountof outer membrane microvesicles (such as OMVs and blebs) produced fromthese PorA⁻PorB⁻RmpM⁻ N. meningitidis. Also disclosed are methods forusing these compositions to induce an immune response to Neisseria, suchas N. meningitidis and N. gonorrhoeae.

Also disclosed are methods for inducing an immune response to N.gonorrhoeae in a mammalian subject. These methods include administeringto the mammalian subject an immunogenic composition comprising aneffective amount of microvesicles from PorA⁻PorB⁻ N. meningitidis and apharmaceutically acceptable carrier, thereby inducing the immuneresponse to N. gonorrhoeae.

The foregoing and other features and advantages of the invention willbecome more apparent from the following detailed description of severalembodiments which proceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A-1B. Construction of outer membrane protein (OMP) deletionmutants. A. Proper deletion of major meningococcal porins PorB and PorAin isogenic OMP deletion strains as confirmed by dot blot withPorB-specific Z15 and PorA-specific P7 monoclonal antibodies (mAbs),respectively (upper panel). rmpM deletion is confirmed by PCR (lowerpanel). rmpM (arrow) indicates the presence of the ˜1.35 kb gene productand chl (arrow), the ˜1.5 kb chloramphenicol resistance cassetteintroduced during double homologous recombination. B. COOMASSIE®-stainedSDS-PAGE gel of OMVs confirms PorA and PorB deletion and reveals nolarge disruption in unrelated protein expression. An increase in PorAfor the PorB single deletion ΔB and the PorB/RmpM double deletion ΔBR isdetectable. ΔA, ΔB, and ΔR are indicative of PorA, PorB, and RmpMdeletion, respectively, where multiple letters indicate multipledeletions. Data are representative of two independent experiments.

FIGS. 2A-2C. OMP deletion diminishes PorB-specific antibody binding andalters the formation of PorB-containing OMP complexes. A. Deletion ofPorA and RmpM decreases the ability of OMVs to inhibit binding ofPorB-specific mAb Z15 to MC58 recombinant PorB (rPorB) in a competitiveELISA (left panel). When fractionated by blue native gel electrophoresis(BNGE) and transferred to a polyvinylidene difluoride (PVDF) membrane,Z15 binds all PorB-containing OMVs, though deletion of rmpM singly (ΔR)or in combination with porA (ΔAR) induces formation of a single dominantPorB-containing complex that is of lower molecular weight (MW) than thewild type (WT) (right panel). B. Of the PorA-containing OMVs, only ARexhibits decreased binding of the PorA-specific P7 mAb in rPorAcompetitive ELISAs (left panel). ΔB and ΔBR are characterized byformation of four large MW PorA-containing complexes (right panel). C.Fractionation of OMVs, followed by visualization with either COOMASSIE®stain (upper panel) or silver stain (lower panel), demonstrates thepresence of unique dominant bands representative of OMP complexes foreach of the deletion mutants. The four porB deletion mutants (ΔB, ΔAB,ΔBR, ΔABR) exhibit the largest alteration in OMP complex bandingprofile. Numbers correlate with complexes analyzed by mass spectrometryin Table 2. Bands visualized by silver stain that are observed in OMVsobtained from multiple OMV types are indicated by arrows. For ELISAs inA and B, data represent the mean±SEM of duplicates of ≥3 independentexperiments. * above each point represents statistical significancerelative to WT OMVs, where # indicates P<0.0001 for binding to all otherOMVs relative to WT. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 bytwo-way ANOVA with Tukey's multiple comparisons post-test. NS=notsignificant. Gels and immunoblots are representative of at least 2independent experiments.

FIGS. 3A-3C. PorB deletion strains exhibit defects in growth. A. Growthcurve of MC58 OMP deletion strains. ΔA and ΔB grow at a rate equivalentto the WT in TSB broth. All other strains exhibit a slight butstatistically significant defect in growth relative to the others.Strains deleted for porB expression produce fewer (B) and smaller (C)colony forming units (CFUs) relative to the other strains. Thedifference in CFU counts is not significant until lateexponential/stationary phase (8 h of growth). Data represent themean±SEM of duplicates of ≥3 independent experiments. ****P<0.0001 bytwo-way ANOVA with Tukey's multiple comparisons post-test. C isrepresentative of two independent experiments.

FIG. 4 . ClustalW (MUSCLE, EMBL-EBI) alignment of the PorB types of theWT MC58 strain (SEQ ID NO: 38) and the isogenic PorA-deficient OChstrain (SEQ ID NO: 39). MC58 and OCh PorB types exhibit amino acidsequence homology in variable L1 and structural L2-L3. Variable L4-L8are heterologous. L1-L8 represent the eight surface-exposed PorB loops.

FIGS. 5A-5D. All OMV antigens induce serum IgG antibodies capable ofbinding multiple meningococcal strains. A. Rabbit immunization schemefor PorA-deficient OMV vaccine study. Pre-immune blood samples weredrawn on the first day of immunization (Day 0) and two weeks followingthe second (Day 28) and third (Day 56) immunization. Samples were alsotaken at the termination of the experiment (Day 84) and assessed for thepresence of meningococcal-specific antibodies relative to pre-immunesamples in whole cell ELISAs (B-D). For B-D, label above the graphindicates strain used to coat whole cell ELISAs. Numeric label (B1-B16)represents the serum sample obtained from each of the 16 immunizedrabbits. Label beneath the bar indicates the immunizing antigen for eachof the sera. BEXSERO® and PBS with aluminum hydroxide adjuvant alonewere used as positive and negative controls, respectively. Datarepresent the mean±SEM of duplicates of ≥3 independent experiments.*P<0.0001 by two-way ANOVA with Tukey's multiple comparisons post-test.

FIG. 6 . Selective OMP deletion enhances cross-protective antibodyresponses. Sera obtained from each of the rabbits immunized with one ofthe six OMV antigens (WT, ΔA, ΔAB, ΔAR, ΔABR, OCh) or the positive(BEXSERO®) or negative (PBS) control antigens were heat-inactivated andassessed for the ability to kill wild type meningococcal strains MC58(black bar), Cu385 (white bar), BB1350 (diagonal bar), Ch501 (horizontalbar), and BB1473 (checkered bar) in the presence of human serum(complement source). The only two antigens that were able to inducebactericidal antibody responses from both of the immunized rabbits thatwere capable of killing all five strains tested were those deleted forboth PorA and PorB expression (ΔAB and ΔABR). Numeric label (B1-B16)represents the serum sample obtained from each of the 16 immunizedrabbits. Label beneath the bar indicates the immunizing antigen for eachof the sera. Hashed line represents threshold of killing (equivalent to2-fold titers), where titers ≥4-fold (those rising above the hashedline) correlate with protective vaccine responses. Titers depictedrepresent titers of serum antibodies from final bleed serum subtractedfor titers of pre-immune serum.

FIGS. 7A-7B. Cross-protective antibody responses correlate withimmunogenicity of high MW proteins. Whole cell lysates of MenB strainsMC58, Cu385, BB1350, Ch501, and BB1473 were fractionated via SDS-PAGEand blotted to nitrocellulose membranes. Lysates were then probed withterminal sera obtained from each of the 16 immunized rabbits to identifyimmunogenic meningococcal proteins. Dominant antigens in WT OMVpreparations (arrows) are also observed to be strongly immunogenic inPorA single mutant antigens (ΔA and OCh) and the BEXSERO® positivecontrol, which, like the WT, expresses PorA, PorB, and RmpM. Serumantibodies that are capable of killing all meningococcal strains testedin bactericidal assays (ΔAB and ΔABR) lack binding to these bands andinstead exhibit binding of high MW bands.

FIG. 8 . Mouse immunization/colonization scheme. Mice were immunizedwith 12.5 μg OMVs/aluminum hydroxide at three fourteen day intervals.Three weeks after the third immunization, mice were administered acocktail of antibiotics to deplete natural flora and β-estradiol tofacilitate staging of the estrous cycle. Animals were thenintravaginally inoculated with ˜1.5×10⁶ CFUs of gonococcal strain F62.Vaginal washes were collected at days 1, 3, 5, and 7 post-inoculation tomonitor CFU counts and vaginal antibodies.

FIGS. 9A-9B. OMV immunization enhances gonococcal clearance in vivo. A.Following immunization, mice were inoculated with gonococcal strain F62and monitored over a period of one week for clearance of colonization.Mice immunized with OCh (open squares) or ΔABR OMVs (closed upwardtriangles) exhibited a statistically significant decrease incolonization density by 7 days post-infection (d.p.i.) as assessed by2-way ANOVA (bold text in table, where P<0.05). n represents sample sizeper group, where uncolonized animals of the initial twenty inoculatedwere not included in the study. B. F62 colonization densities forindividual mice throughout the course of the study. By 7 d.p.i., themedian density of colonization for OCh OMV- and ΔABR OMV-immunized miceis equivalent to 0 CFU/ml, while median density for MC58 OMV-immunizedanimals is 60 CFU/ml. Alum-immunized and unimmunized controls remainlargely colonized. Bars represent median±interquartile range ofcolonization density. *P<0.05 and **P<0.01 by Mann-Whitney U test.

FIG. 10 . Antibodies present in pooled sera from OCh OMV- and ΔABROMV-immunized mice bind to unique high MW proteins that are not bound byserum antibodies from MC58 OMV-immunized mice. Whole cell lysates ofgonococcal strains FA19, FA1090, MS11, and F62 were fractionated bySDS-PAGE and blotted to nitrocellulose. Lysates of the parentalmeningococcal strain used to create the OMV antigens, MC58, was alsofractionated as a control. Sera were pooled from all twenty animals ineach group following the third immunization and were used to probe thelysates for binding of serum antibodies. Serum antibodies from OCh OMV-and ΔABR OMV-immunized animals bound unique ˜70 kDa and ˜90 kDa proteinsthat were not bound by sera from MC58 OMV-immunized animals (arrows).Serum antibodies from aluminum hydroxide-immunized control miceexhibited no binding.

FIGS. 11A-11B. ClustalW (MUSCLE, EMBL-EBI) analysis of the producedchimeric PorB types. L1-L8 represent surface-expressed loops. Shown areOCh (SEQ ID NO: 39), M(1-4)C(5-8)(SEQ ID NO: 40), M(1-6)C(7-8) (SEQ IDNO: 41), M(1-4)C(5-6)M(7-8) (SEQ ID NO: 42), C(1-4)M(5-8) (SEQ ID NO:43), C(1-6)M(7-8)(SEQ ID NO: 44), C(1-4)M(5-6)C(7-8) (SEQ ID NO: 45),M(1-4)B(5-8) (SEQ ID NO: 46), M(1-6)B(7-8) (SEQ ID NO: 47),M(1-4)B(5-6)M(7-8) (SEQ ID NO: 48), B(1-4)M(5-8) (SEQ ID NO: 49),B(1-6)M(7-8) (SEQ ID NO: 50), and B(1-4)M(5-6)B(7-8) (SEQ ID NO: 51).The N-terminal portion of each sequence is shown in panel A, and theC-terminal portion of the sequence is shown in panel B.

SEQUENCE LISTING

The nucleic and amino acid sequences listed herein are shown usingstandard letter abbreviations for nucleotide bases, and one letter codefor amino acids, as defined in 37 C.F.R. 1.822. Only one strand of eachnucleic acid sequence is shown, but the complementary strand isunderstood as included by any reference to the displayed strand. TheSequence Listing is submitted as an ASCII text file in the form of thefile named “9531-100847-07_Sequence_Listing.xml,” 69,236 bytes, whichwas created on Aug. 31, 2022, which is incorporated by reference herein.In the accompanying sequence listing:

SEQ ID NOS: 1-37 are the nucleic acid sequence of primers.

SEQ ID NOS: 38-51 are the amino acid sequences of PorB proteins.

DETAILED DESCRIPTION OF SEVERAL EMBODIMENTS

N. meningitidis is a Gram-negative bacterium and the causative agent ofmeningococcal meningitis and septicemia. Its only known host is thehuman, and it may be carried asymptomatically by approximately 10% ofthe population (Caugant et al, 1994, J. Clin. Microbiol. 32 323). N.meningitidis can express a polysaccharide capsule, and this allowsclassification of the bacteria according to the nature of the capsuleexpressed. There are at least twelve serogroups of N. meningitidis: A,B, C, 29-E, H, I, K, L, W135, X, Y, and Z, of which serogroups A, B, C,Y, and W cause 90% of meningococcal disease (Poolman et al. (1983)Infect Immun 40: 398-406, Infect. Agents and Dis. 4 13). Capsularpolysaccharide vaccines directed against serogroups A, C, Y, and W areavailable; subcapsular vaccines for prevention of serogroup B diseaseare available but do not target all serotypes and do not providecross-protection against N. gonorrhoeae.

Disclosed herein are compositions that include isolated PorA⁻PorB⁻RmpM⁻N. meningitidis. The isolated PorA⁻PorB⁻RmpM⁻ N. meningitidis can beserogroup A, B, or C, or any other serotype Immunogenic compositions canbe produced that include outer membrane microvesicles from thesePorA⁻PorB⁻RmpM⁻ N. meningitidis, such as blebs and/or OMVs, and apharmaceutically acceptable carrier. Optionally, these pharmaceuticalcarriers can also include an adjuvant. Optionally, the microvesicles caninclude a heterologous protein.

It is also disclosed herein that pharmaceutical compositions including aPorA⁻PorB⁻ Neisseria meningitidis, such as a PorA⁻PorB⁻RmpM⁻ N.meningitidis, are of use in inducing an immune response in a subject.The immune response can be a protective immune response or a therapeuticimmune response.

In some embodiments, the subject has a N. meningitidis infection, andadministration of the immunogenic composition increases clearance of theNeisseria meningitidis. In other embodiments, the subject has a N.gonorrhoeae infection, and administration of the immunogenic compositionincreases clearance of N. gonorrhoeae. In further embodiments, themammalian subject is a healthy subject. In additional embodiments, themammalian subject is a human In some embodiments, the immune response isa protective immune response. In other embodiments, the immune responseis a therapeutic response.

In more embodiments, methods are disclosed for including an immuneresponse to Neisseria gonorrhoeae in a mammalian subject. These methodsinclude administering to the mammalian subject an immunogeniccomposition comprising an effective amount of outer membranemicrovesicles from PorA⁻PorB⁻ N. meningitidis and a pharmaceuticallyacceptable carrier, thereby inducing the immune response to Neisseriagonorrhoeae. In some embodiments, the PorA⁻PorB⁻ N. meningitidis isRmpM⁻. The microvesicles can be outer membrane vesicles, blebs, or acombination thereof. In specific non-limiting examples, the N.meningitidis is serogroup A, B, or C. Optionally, the immunogeniccomposition further includes an adjuvant.

In some non-limiting examples, the subject has a Neisseria gonorrhoeaeinfection, and administration of the immunogenic composition increasesclearance of the Neisseria gonorrhoeae. In other non-limiting examples,the mammalian subject does not have an infection with Neisseriagonorrhoeae or Neisseria meningitidis. In any of the disclosed methods,the mammalian subject can be human.

In other embodiments, compositions are disclosed herein that includegenetically modified PorB that are chimeric recombinant combinations oftwo or more PorB from N. meningitidis. Exemplary chimeric recombinantPorB amino acid sequences are shown in FIGS. 11A-11B. Nucleic acidmolecules can be produced that encode these chimeric recombinant PorB.An isolated N. meningitidis that includes one or more of these proteinsis PorB⁺. An isolated PorA⁻PorB⁻ N. meningitidis does not include one ofthese chimeric recombinant PorB proteins.

Summary of Terms

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes X, published by Jones & BartlettPublishers, 2009; and Meyers et al. (eds.), The Encyclopedia of CellBiology and Molecular Medicine, published by Wiley-VCH in 16 volumes,2008; and other similar references. As used herein, the singular forms“a,” “an,” and “the,” refer to both the singular as well as plural,unless the context indicates otherwise. For example, the term “anantigen” includes single or plural antigens and can be consideredequivalent to the phrase “at least one antigen.” As used herein, theterm “comprises” means “includes.” It is further to be understood thatany and all base sizes or amino acid sizes, and all molecular weight ormolecular mass values, given for nucleic acids or polypeptides areapproximate, and are provided for descriptive purposes, unless otherwiseindicated. Although many methods and materials similar or equivalent tothose described herein can be used, particular suitable methods andmaterials are described below. In case of conflict, the presentspecification, including explanations of terms, will control. Inaddition, the materials, methods, and examples are illustrative only andnot intended to be limiting. To facilitate review of the variousembodiments, the following explanations of terms are provided:

Administration: The introduction of a composition into a subject by achosen route. Administration can be local or systemic. For example, ifthe chosen route is intranasal, the composition is administered byintroducing the composition into the nasal passages of the subject.

Similarly, if the chosen route is intramuscular, the composition isadministered by introducing the composition into a muscle of thesubject. If the chosen route is oral, the composition is administered byintroducing the subject ingesting the composition. Exemplary routes ofadministration of use in the methods disclosed herein include, but arenot limited to, oral, injection (such as subcutaneous, intramuscular,intradermal, intraperitoneal, and intravenous), sublingual, rectal,transdermal (for example, topical), intranasal, vaginal, and inhalationroutes.

Amino acid substitution: The replacement of an amino acid in apolypeptide with one or more different amino acids. In the context of aprotein sequence, an amino acid substitution is also referred to as amutation.

Antibody: An immunoglobulin, antigen-binding fragment, or derivativethereof, that specifically binds and recognizes an analyte (antigen)such as an antigen on a microvesicle (such as an outer membrane vesicle(OMV) or bleb) of Neisseria meningitidis. The term “antibody” is usedherein in the broadest sense and encompasses various antibodystructures, including but not limited to monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredantigen-binding activity. Non-limiting examples of antibodies include,for example, intact immunoglobulins and variants and fragments thereofthat retain binding affinity for the antigen. Examples of antibodyfragments include but are not limited to Fv, Fab, Fab′, Fab′-SH,F(ab′)₂; diabodies; linear antibodies; single-chain antibody molecules(e.g. scFv); and multispecific antibodies formed from antibodyfragments. Antibody fragments include antigen binding fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies (see, e.g., Kontermann andDubel (Ed), Antibody Engineering, Vols. 1-2, 2^(nd) Ed., Springer Press,2010).

Chimeric Protein: A chimeric protein, also called a hybrid protein, thatincludes portions of the protein from two different sources. A chimericPorB includes portions of the PorB from two different sources, such as aportion from N. meningitidis and a portion from N. gonorrhoeae, or aportion from two different serogroups, such as serogroup A, B, C, Y, andW-135. In one embodiment, the different portions can be N-terminal andC-terminal. In other embodiments, specific domains, such as loops, aresubstituted by the protein from a different source.

Control: A reference standard. In some embodiments, the control is anegative control sample obtained from a healthy patient. In otherembodiments, the control is a positive control sample obtained from apatient immunized with a microvesicle (such as an outer membrane vesicle(OMV) or bleb) of Neisseria meningitidis. In still other embodiments,the control is a historical control or standard reference value or rangeof values (such as a previously tested control sample, such as a groupof patients with known prognosis or outcome, or group of samples thatrepresent baseline or normal values).

A difference between a test sample and a control can be an increase orconversely a decrease. The difference can be a qualitative difference ora quantitative difference, for example a statistically significantdifference. In some examples, a difference is an increase or decrease,relative to a control, of at least about 5%, such as at least about 10%,at least about 20%, at least about 30%, at least about 40%, at leastabout 50%, at least about 60%, at least about 70%, at least about 80%,at least about 90%, at least about 100%, at least about 150%, at leastabout 200%, at least about 250%, at least about 300%, at least about350%, at least about 400%, at least about 500%, or greater than 500%.

Degenerate variant: In the context of the present disclosure, a“degenerate variant” refers to a polynucleotide encoding a polypeptidethat includes a sequence that is degenerate as a result of the geneticcode. There are 20 natural amino acids, most of which are specified bymore than one codon. Therefore, all degenerate nucleotide sequencesencoding a peptide are included as long as the amino acid sequence ofthe peptide encoded by the nucleotide sequence is unchanged.

Effective amount: An amount of agent, such as an immunogen, such as a N.meningitidis microvesicle, that is sufficient to elicit a desiredresponse, such as an immune response in a subject. It is understood thatto obtain a protective immune response against an organism of interestcan require multiple administrations of a disclosed immunogen, and/oradministration of a disclosed immunogen as the “prime” in a prime boostprotocol wherein the boost immunogen can be different from the primeimmunogen. Accordingly, an effective amount of a disclosed immunogen canbe the amount of the immunogen sufficient to elicit a priming immuneresponse in a subject that can be subsequently boosted with the same ora different immunogen to elicit a protective immune response.

In one example, a desired response is to inhibit or reduce or prevent aNeisseria gonorrhoeae or Neisseria meningitidis infection. The Neisseriagonorrhoeae or N. meningitidis infection does not need to be completelyeliminated or reduced or prevented for the method to be effective. Forexample, administration of an effective amount of the agent can decreasethe Neisseria gonorrhoeae or N. meningitidis infection (for example, asmeasured by bacteria number or by number or percentage of subjectsinfected by Neisseria gonorrhoeae or Neisseria meningitidis) by adesired amount, for example by at least 50%, at least 60%, at least 70%,at least 80%, at least 90%, at least 95%, at least 98%, or even at least100% (elimination or prevention of detectable hPIV infection), ascompared to a suitable control.

Epitope: An antigenic determinant These are particular chemical groupsor peptide sequences on a molecule that are antigenic, such that theyelicit a specific immune response, for example, an epitope is the regionof an antigen to which B and/or T cells respond. An antibody can bind toa particular antigenic epitope, such as an epitope presented on amicrovesicle of Neisseria gonorrhoeae or Neisseria meningitidis.Epitopes can be formed both from contiguous amino acids or noncontiguousamino acids juxtaposed by tertiary folding of a protein.

Heterologous: Originating from a different genetic source, so that thebiological components that are not found together in nature. Thecomponents may be host cells, genes, or regulatory regions, such aspromoters. Although the heterologous components are not found togetherin nature, they can function together, as when a promoter heterologousto a gene is operably linked to the gene. Another example is where aNeisserial sequence is heterologous to a Neisserial host of a differentstrain. “Heterologous” as used herein in the context of proteinsexpressed in two different bacterial strains, e.g., “heterologous PorA.”

Immune response: A response of a cell of the immune system, such as a Bcell, T cell, or monocyte, to a stimulus, such as Neisseria. In oneembodiment, the response is specific for a particular antigen (an“antigen-specific response”). In one embodiment, an immune response is aT cell response, such as a CD4+ response or a CD8+ response. In anotherembodiment, the response is a B cell response, and results in theproduction of specific antibodies. A “protective immune response” is animmune response that confers protection against a disease caused by amember of Neisseria, such as N. meningitidis serogroups, particularlyserogroups A, B, C, Y, and W-135, and/or Neisseria gonorrhoeae. A“therapeutic immune response” treats an existing infection withNeisseria. In some embodiments, the subject has a N. meningitidisinfection, and administration of the immunogenic composition increasesclearance of the Neisseria meningitidis. In other embodiments, thesubject has a Neisseria gonorrhoeae infection, and administration of theimmunogenic composition increases clearance of Neisseria gonorrhoeae.

Immunogen: A compound, composition, or substance (for example, acomposition including outer membrane microvesicles from PorA⁻PorB⁻Neisseria meningitidis) that can elicit an immune response in an animal,including compositions that are injected or absorbed into an animal.Administration of an immunogen to a subject can lead to immunity againsta pathogen of interest, such as Neisseria gonorrhoeae and/or Neisseriameningitidis.

Immunogenic composition: A composition comprising outer membranemicrovesicles from PorA⁻PorB⁻ N. meningitidis that induces a measurableCTL response against Neisseria gonorrhoeae and/or Neisseriameningitidis, or induces a measurable B cell response (such asproduction of antibodies) against Neisseria gonorrhoeae and/or Neisseriameningitidis, when administered to a subject. For in vivo use, theimmunogenic composition will typically include outer membranemicrovesicles from PorA⁻PorB⁻ N. meningitidis in a pharmaceuticallyacceptable carrier and optionally may also include other agents, such asan adjuvant. The phrase “in an effective amount to elicit an immuneresponse” means that there is a detectable difference between an immuneresponse indicator measured before and after administration of aparticular immunogenic composition. Immune response indicators includebut are not limited to: antibody titer or specificity, as detected by anassay such as enzyme-linked immunosorbent assay (ELISA), bactericidalassay, flow cytometry, immunoprecipitation, Ouchterlony immunodiffusion;binding detection assays of, for example, spot, western blot or antigenarrays; cytotoxicity assays, etc.

Inhibiting or treating a disease: Inhibiting the full development of adisease or condition, for example, in a subject who is at risk for adisease such as a Neisseria gonorrhoeae and/or N. meningitidisinfection. “Treatment” refers to a therapeutic intervention thatameliorates a sign or symptom of a disease or pathological conditionafter it has begun to develop. The term “ameliorating,” with referenceto a disease or pathological condition, refers to any observablebeneficial effect of the treatment. Inhibiting a disease can includepreventing or reducing the risk of the disease, such as preventing orreducing the risk of bacterial infection. The beneficial effect can beevidenced, for example, by a delayed onset of clinical symptoms of thedisease in a susceptible subject, a reduction in severity of some or allclinical symptoms of the disease, a slower progression of the disease, areduction in the bacterial load, an improvement in the overall health orwell-being of the subject, or by other parameters that are specific tothe particular disease. A “prophylactic” treatment is a treatmentadministered to a subject who does not exhibit signs of a disease orexhibits only early signs for the purpose of decreasing the risk ofdeveloping pathology.

Isolated: An “isolated” biological component has been substantiallyseparated or purified away from other biological components, such asother biological components in which the component naturally occurs,such as other chromosomal and extrachromosomal DNA, RNA, membranes,cells and proteins. Microvesicles that have been “isolated” includethose purified by standard purification methods. Isolated does notrequire absolute purity, and can include microvesicles that are at least50% isolated, such as at least 75%, 80%, 90%, 95%, 98%, 99%, or even99.9% isolated from other components of the bacteria that product them.

Microvesicles: Outer membrane microvesicles are produced from the outermembrane of Neisseria, and include both outer membrane vesicles (OMVs)and blebs. OMVs are produced by solubilizing Neisseria in a processwherein the outer membranes form vesicles. Blebs are produced by buddingfrom living Neisseria. Methods from the production of outer membranemicrovesicles are known in the art, see for example, van de Waterbeemdet al., PLOS One, doi.org/10.1371/journal.pone.0065157, May 31, 2013).OMV and blebs can be purified from intact Neisseria, and are generally100 to 300 nm.

Outer Membrane Protein Reduction Modifiable Protein (Rmp)M: Aperiplasmic protein from N. meningitidis that comprises an N-terminaldomain (residues 1-47) and a separate globular C-terminal domain(residues 65-219) responsible for binding to peptidoglycan. TheN-terminal fragment of RmpM binds to both the major outer membraneporins, PorA and PorB. Analysis by semi-native SDS-PAGE established thatboth recombinant full-length RmpM and an N-terminal fragment weresufficient to stabilize the PorA and PorB oligomeric complexes. Themeso-diaminopimelate moiety plays a role in peptidoglycan recognition byRmpM. Site-directed mutagenesis showed that two highly conservedresidues, Asp120 and Arg135, play a role in peptidoglycan binding, seeMaharjan et al., Microbiology 162: 364-375, 2016. See also Li et al.,PLOS Biology, doi.org/10.1371/journal.pone.0090525, Mar. 4, 2014 andGENBANK Accession No. X05105.1, as available on Jun. 30, 2018,incorporated herein by reference. The yield of OMV is higher in a N.meningitidis strain expressing a truncated N-terminal fragment of RmpM(ΔC-term RmpM) than in a wild-type strain. This strain is also RpmM− asit does not produce funcational RmpM protein. Generally, a RmpM⁻Neisseria (also called “ΔR”) does not produce functional RmpM protein.

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 19th Edition, 1995,describes compositions and formulations suitable for pharmaceuticaldelivery of the disclosed immunogens.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol, or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically neutral carriers, pharmaceuticalcompositions (such as immunogenic compositions) to be administered cancontain minor amounts of non-toxic auxiliary substances, such as wettingor emulsifying agents, preservatives, and pH buffering agents and thelike, for example sodium acetate or sorbitan monolaurate. In particularembodiments, suitable for administration to a subject the carrier may besterile, and/or suspended or otherwise contained in a unit dosage formcontaining one or more measured doses of the composition suitable toinduce the desired immune response. It may also be accompanied bymedications for its use for treatment purposes. The unit dosage form maybe, for example, in a sealed vial that contains sterile contents or asyringe for injection into a subject, or lyophilized for subsequentsolubilization and administration or in a solid or controlled releasedosage.

Polypeptide: Any chain of amino acids, regardless of length orpost-translational modification (e.g., glycosylation orphosphorylation). “Polypeptide” applies to amino acid polymers includingnaturally occurring amino acid polymers and non-naturally occurringamino acid polymer as well as in which one or more amino acid residue isa non-natural amino acid, for example, an artificial chemical mimetic ofa corresponding naturally occurring amino acid. A “residue” refers to anamino acid or amino acid mimetic incorporated in a polypeptide by anamide bond or amide bond mimetic. A polypeptide has an amino terminal(N-terminal) end and a carboxy terminal (C-terminal) end. “Polypeptide”is used interchangeably with peptide or protein, and is used herein torefer to a polymer of amino acid residues.

Porin (Por)A: PorA is a Neisseria porin. PorA monomer topology showseight extracellular loops (Derrick et al., 1999, Infect. Immun 672406-13; van der Ley et al., 1991, Infect. Immun 59 2963) in theprotein. The longest loops (1 and 4) are the most variable, hence arereferred to as Variable Region 1 (VR1) and Variable Region 2 (VR2). Lessvariability is seen in loops 5 and 6 (also called semi-variable SVR1 and2, or variable region 3 and 4, respectively), with essentially novariability in the remaining loops. Loop 3 is predicted to form a “plug”in the pore formed by each subunit of the PorA trimer. Even within VR1and VR2, most of the variability is confined to residues predicted toform the tip of each loop. Indeed, in both mice and in immunized humanvolunteers, epitope mapping showed that the majority of the antibodyresponse is directed at the “top” of loops 1 and 4, the region that isvariable between strains (van der Voort, et al., 1997, FEMS Immunol.Med. Microbiol. 17 139-48).

This protein generates an immune response in both patients andasymptomatic carriers, to the extent that it has been used as a markerfor strain identification, representing the serosubtype system(McGuinness et al., 1990, J Exp Med. 171 1871-82). PorA has been used ineffective and registered vaccine formulations and elicits effectivebactericidal antibodies. However, strain-to-strain variability insurface loops results in a variable target, and vaccines are typicallyPorA type-specific. Efforts have been made to generate multivalent PorAvaccines covering up to six different PorA types (van der Voort et al.,1996, Infect Immun. 64 2745-51). A PorA⁻ Neisseria (also called ΔA) doesnot produce functional PorA protein.

Porin B (PorB): A 16-pass transmembrane protein from Neisseria that is aporin and forms a β-barrel structure with eight surface-exposed loops(L1-L8) (Tanabe et al. (2010) Proc Natl Acad Sci USA 107: 6811-6816).Two of these, L2 and L3, are structural and do not vary considerably inamino acid sequence among different MenB strains. The remaining six, L1and L4-L8, undergo antigenic variation; it is the binding of antibodiesto these loops that forms the basis for meningococcal serotyping (Fraschet al. (1985) Rev Infect Dis 7: 504-510). A PorB⁻ Neisseria (also calledΔB) does not produce functional PorB protein.

Prime-boost vaccination: An immunotherapy including administration of afirst immunogenic composition (the primer vaccine) followed byadministration of another immunogenic composition (the booster vaccine)to a subject to induce an immune response. The primer vaccine and/or thebooster vaccine are immunogens to which the immune response is directed.The booster vaccine is administered to the subject after the primervaccine; a suitable time interval between administration of the primervaccine and the booster vaccine, and examples of such timeframes aredisclosed herein. In some embodiments, the primer vaccine, the boostervaccine, or both primer vaccine and the booster vaccine additionallyinclude an adjuvant

Recombinant: A recombinant nucleic acid molecule is one that has asequence that is not naturally occurring, for example, includes one ormore nucleic acid substitutions, deletions or insertions, and/or has asequence that is made by an artificial combination of two otherwiseseparated segments of sequence. This artificial combination can beaccomplished by chemical synthesis or, more commonly, by the artificialmanipulation of isolated segments of nucleic acids, for example, bygenetic engineering techniques.

A recombinant protein is one that has a sequence that is not naturallyoccurring or has a sequence that is made by an artificial combination oftwo otherwise separated segments of sequence. In several embodiments, arecombinant protein is encoded by a heterologous (for example,recombinant) nucleic acid that has been introduced into a host cell,such as a bacterial or eukaryotic cell, or into the genome of arecombinant virus.

Sequence identity: The similarity between amino acid sequences isexpressed in terms of the similarity between the sequences, otherwisereferred to as sequence identity. Sequence identity is frequentlymeasured in terms of percentage identity; the higher the percentage, themore similar the two sequences are. Homologs, orthologs, or variants ofa polypeptide will possess a relatively high degree of sequence identitywhen aligned using standard methods.

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. In the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

Variants of a polypeptide are typically characterized by possession ofat least about 75%, for example, at least about 80%, 85%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity counted over thefull-length alignment with the amino acid sequence of interest. Proteinswith even greater similarity to the reference sequences will showincreasing percentage identities when assessed by this method, such asat least 80%, at least 85%, at least 90%, at least 95%, at least 98%, orat least 99% sequence identity. When less than the entire sequence isbeing compared for sequence identity, homologs and variants willtypically possess at least 80% sequence identity over short windows of10-20 amino acids, and may possess sequence identities of at least 85%or at least 90% or 95% depending on their similarity to the referencesequence. Methods for determining sequence identity over such shortwindows are available at the NCBI website on the internet.

As used herein, reference to “at least 90% identity” (or similarlanguage) refers to “at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99%, or even 100% identity” to a specified referencesequence.

Serogroup: Classification, such as of Neisseria meningitidis by virtueof immunologically detectable variations in the capsular polysaccharide.About 12 serogroups are known: A, B, C, X, Y, Z, 29-E, W-135, H, I, K,and L. Any one serogroup can encompass multiple serotypes and multipleserosubtypes. A serotype is classification of Neisseria meningitidisstrains based on monoclonal antibody-defined antigenic differences inthe outer membrane protein porn PorB, or upon VR typing of amino acidsequences deduced from DNA sequencing. A single serotype can be found inmultiple serogroups and multiple serosubtypes. “Serosubtype” isclassification of Neisseria meningitidis strains based onantibody-defined antigenic variations on the outer membrane protein pornPorA, or upon VR typing of amino acid sequences deduced from DNAsequencing (Sacchi et al., 2000, J. Infect. Dis. 182:1169; see also theMulti Locus Sequence Typing web site). Most variability between PorAproteins occurs in two (loops I and IV) of eight putative,surface-exposed loops. The variable loops I and IV have been designatedVR1 and VR2, respectively. A single serosubtype can be found in multipleserogroups and multiple serotypes.

Subject: Living multi-cellular vertebrate organisms, a category thatincludes human and non-human mammals. In an example, a subject is ahuman. In an additional example, a subject is selected that is in needof inhibiting of a Neisseria infection. For example, the subject iseither uninfected and at risk for infection, or is infected in need oftreatment.

Under conditions sufficient for: A phrase that is used to describe anyenvironment that permits a desired activity.

Vaccine: A preparation of immunogenic material capable of stimulating animmune response, administered for the prevention, amelioration, ortreatment of infectious or other types of disease. The immunogenicmaterial may include attenuated or killed microorganisms (such asbacteria or viruses), or antigenic proteins, peptides, or DNA derivedfrom them. A vaccine may include a disclosed immunogen, such as outermembrane microvesicles from PorA⁻PorB⁻ Neisseria meningitidis, such as aPorA⁻PorB⁻RmpM⁻ Neisseria meningitidis. Vaccines can elicit bothprophylactic (preventative or protective) and therapeutic responses.Methods of administration vary according to the vaccine, but may includeinoculation, ingestion, inhalation, or other forms of administration.Vaccines may be administered with an adjuvant to boost the immuneresponse. In one specific, non-limiting example, a vaccine preventsand/or reduces the severity of the symptoms associated with hPIVinfection and/or decreases the viral load compared to a control.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including explanations ofterms, will control. In addition, the materials, methods, and examplesare illustrative only and not intended to be limiting.

Neisseria Strains and Outer Membrane Microvesicles

In some embodiments, isolated PorA⁻PorB⁻RmpM⁻ N. meningitidis aredisclosed herein. These N. meningitidis are not naturally occurring, asPorB⁻ N. meningitidis do not occur in nature. Isolated PorA⁻PorB⁻ N.meningitidis, such as isolated PorA⁻PorB⁻RmpM⁻ Neisseria meningitidis,are of use in the disclosed methods.

Generally, PorA⁻PorB⁻RmpM⁻ N. meningitidis are deficient from theproduction of PorA, PorB, and RmpM. Thus, these PorA, PorB, and RmpMsubcapsular proteins cannot be detected in the surface of these N.meningitidis. Thus, PorA, PorB, and Rpm do not function in these N.meningitidis.

In some embodiments, there is a deletion in the PorA, PorB, and/or RmpMgenes in these N. meningitidis, so that the corresponding protein is notproduced. In other embodiments, there is a stop codon inserted in thePorA, PorB, and/or RmpM gene(s), so that the corresponding protein isnot produced. In other embodiments, the genes include a mutation suchthat the protein is not functional or immunogenic, e.g., the protein notpresent on the outer membrane of the N. meningitidis.

In some embodiments, production of these surface proteins is decreasedby at least 80%, 85%, 90%, 95%, 98%, 99% or is complete absent.Generally, in the strains of use in the methods disclosed herein, PorA,PorB, and RmpM is not detectable on the bacterial surface. Suitablemethods for detecting PorA, PorB, and RmpM on the cell surface includewhole cell ELISA using monoclonal and polyclonal protein-specificantibodies.

Modified strains can be generated by recombination techniques, and bynon-recombinant techniques such as, for example, exposure to chemicals,radiation, or other DNA modifying or damaging agent, and the like.Modified strains having a desired protein expression profile,specifically wherein PorA, PorB, and/or RmpM is not detectable at thecell surface, can be identified through screening.

The N. meningitidis can be any type. N. meningitidis strains can bedivided into serologic groups, serotypes, and subtypes on the basis ofreactions with polyclonal (Frasch et al. (1985) Rev Infect Dis 7:504-510, C. E. and Chapman, 1973, J. Infect. Dis. 127: 149-154) ormonoclonal antibodies that interact with different surface antigens.Serogroup is based on immunologically detectable variations in thecapsular polysaccharide. About 12 serogroups (A, B, C, X, Y, Z, 29-E,and W-135) are known. In some embodiments, the PorA⁻PorB⁻RmpM⁻ N.meningitidis are serogroup A, B, or C.

N. meningitidis also can be divided into clonal groups or subgroups,using various techniques that directly or indirectly characterize thebacterial genome. These techniques include multilocus enzymeelectrophoresis (MLEE), based on electrophoretic mobility variation ofan enzyme, which reflects the underlying polymorphisms at a particulargenetic locus. By characterizing the variants of a number of suchproteins, genetic “distance” between two strains can be inferred fromthe proportion of mismatches. Similarly, clonality between two isolatescan be inferred if the two have identical patterns of electrophoreticvariants at a number of loci. Multilocus sequence typing (MLST) can alsobe used to characterize the microorganisms. Using MLST, the geneticdistance between two isolates, or clonality, is inferred from theproportion of mismatches in the DNA sequences of 11 housekeeping genesin N. meningitidis strains (Maiden et al., 1998, Proc. Natl. Acad. Sci.USA 95:3140). Any strain can be selected and used to produce aPorA⁻PorB⁻ N. meningitidis, such as a PorA⁻PorB⁻RmpM⁻ N. meningitidis.

Isolated N. meningitidis can be transformed to express a heterologousprotein. These recombinant N. meningitidis are also of use in themethods disclosed herein, provided they are PorA⁻PorB⁻RmpM⁻. Theisolated N. meningitidis can be transformed to express additionalantigens such as those exemplified in PCT Publication Nos. WO 99/24578,WO 99/36544; WO 99/57280, WO 00/22430, and WO 00/66791, as well asantigenic fragments of such proteins.

In some embodiments, outer membrane microvesicles are produced fromPorA⁻PorB⁻RmpM⁻ N. meningitidis. It is disclosed herein that aneffective amount of microvesicles from a PorA⁻PorB⁻ N. meningitidis,such as an effective amount of microvesicles from a PorA⁻PorB⁻RmpM⁻ N.meningitidis can be used to induce an immune response to Neisseria, suchas to N. meningitidis and/or N. gonorrhoeae. Immunogenic compositionscan be produced including an effective amount of microvesicles from aPorA⁻PorB⁻RmpM⁻ N. meningitidis and a pharmaceutically acceptablecarrier. In some embodiments, outer membrane microvesicles from thePorA⁻PorB⁻RmpM⁻ N. meningitidis that expresses a heterologous protein.These vesicles do not include the PorA, PorB, or RmpM, but include theheterologous protein.

The microvesicles can be outer membrane vesicles, blebs, or acombination thereof. Blebs are budded from living N. meningitidis.Methods for isolating blebs are also known in the art, see for example,Post et al., J. Biological Chem. 280: 38383-38394, 2005, incorporatedherein by reference. OMV are produced by solubilizing Neisseria. Methodsof producing OMV are disclosed, for example, in U.S. Published PatentApplication No. 2012/0328643, incorporated herein by reference. Methodsfor the preparation of OMVs also are disclosed in Claassen et al.(Vaccine (1996) 14:1001-1008); Cartwright et al. (Vaccine (1999)17:2612-2619); Peeters et al. (Vaccine (1996) 14:1009-1015); Fu et al.(Biotechnology NY (1995) 12:170-74); Davies et al. (J. Immunol. Meth.(1990) 134:215-225); Saunders et al. (Infect. Immun. (1999) 67:113-119);Draabick et al. (Vaccine (2000) 18:160-172); Moreno et al. (Infect.Immun. (1985) 47:527-533); Milagres et al. (Infect. Immun. (1994)62:4419-4424); Naess et al. (Infect. Immun. (1998) 66:959-965];Rosenqvist et al. [Dev. Biol. Stand. (1998) 92:323-333]; Haneberg et al.[Infect. Immunn. (1998) 66:1334-41); Andersen et al. (Vaccine (1997)15:1225-34); and Bjune et al. (Lancet (1991) 338:1093-96).

In some embodiments, OMVs are prepared by deoxycholate extraction. Anextraction protocol is disclosed in Fredriksen et al., (1991) NIPH Ann.14(2):67-79. Additional methods are disclosed, for example, in, Bjune etal. (Lancet (1991) 338(8775):1093-96), Fredriksen et al. (1991) NIPHAnnals 14: 67-79, Pages 818-824 of Pathobiology and immunobiology ofNeisseriaceae (eds. Conde-Glez et al.) ISBN 968-6502-13-0). The OMV (forexample, as obtained by deoxycholate extraction) can be treated toremove certain components. For instance, pyrogens or toxic componentsmay be removed (e.g. LOS).

Immunogenic Compositions and Methods of Use

Immunogenic Compositions of use in the disclosed method include outermembrane microvesicles from PorA⁻PorB⁻ N. meningitidis, such asPorA⁻PorB⁻RmpM⁻ N. meningitidis. The microvesicles can be OMV and/orblebs. The immunogenic compositions of use in the disclosed methods caninclude a mixture of microvesicles (e.g., OMV and blebs), whichmicrovesicles can be from the same or different strains. In anotherembodiment, the immunogenic compositions can comprise a mixture ofvesicles from 2, 3, 4, 5 or more PorA⁻PorB⁻ N. meningitidis strains,such as PorA⁻PorB⁻RmpM⁻ strains, where the vesicles can be OMV, blebs orboth.

Optionally, an immunogenic composition can include an adjuvant.Adjuvants can include a suspension of minerals (alum, aluminumhydroxide, or phosphate) on which antigen is adsorbed; or water-in-oilemulsion in which antigen solution is emulsified in mineral oil (forexample, Freund's incomplete adjuvant), sometimes with the inclusion ofkilled mycobacteria (Freund's complete adjuvant) to further enhanceantigenicity Immunostimulatory oligonucleotides (such as those includinga CpG motif) can also be used as adjuvants (for example, see U.S. Pat.Nos. 6,194,388; 6,207,646; 6,214,806; 6,218,371; 6,239,116; 6,339,068;6,406,705; and 6,429,199). Adjuvants also include biological molecules,such as costimulatory molecules. Exemplary biological adjuvants includeinterleukin (IL)-2, IL-12, RANTES, granulocyte macrophage colonystimulating factor (GM-CSF), tumor necrosis factor (TNF)-α, andinterferon (IFN)-γ.

In one embodiment, the microvesicles, such as the OMV and/or blebs, canused with an aluminum hydroxide adjuvant. One protein:adjuvant ratio is1:67 (wt/wt). However, other ratios can be used, such as 1:20, 1:30,1:40, 1:50, 1:33, 1;60, 1:65, 1:70, 1:75, 1:80 or 1:85).

Additional adjuvants include Complete Freund's Adjuvant, IncompleteFreund's Adjuvant, Gerbu adjuvant (GMDP; C.C. Biotech Corp.), RIBI fowladjuvant (MPL; RIBI Immunochemical Research, Inc.), potassium alum,aluminum phosphate, QS21 (Cambridge Biotech), Titer Max adjuvant(CytRx), and Quil A adjuvant. Exogenous lipopolysaccharide (LPS) canalso be used as an adjuvant.

The immunogenic compositions also can also include other agents, such asbinders. Binders include, but are not limited to,carboxymethylcellulose, ethyl cellulose, microcrystalline cellulose, orgelatin; excipients such as starch, lactose or dextrins, disintegratingagents such as alginic acid, sodium alginate, Primogel, corn starch andthe like; lubricants such as magnesium stearate or Sterotex; glidantssuch as colloidal silicon dioxide; sweetening agents such as sucrose orsaccharin, a flavoring agent such as peppermint, methyl salicylate ororange flavoring, and a coloring agent. The compositions can alsoinclude gum arabic, syrup, lanolin, starch, etc., that forms a vehiclefor delivery. Included are substances that, in the presence ofsufficient liquid, impart to a composition the adhesive quality neededfor the preparation of pills or tablets.

Exemplary “pharmaceutically acceptable carriers” include liquid carriers(such as water, saline, culture medium, aqueous dextrose, and glycols)and solid carriers (such as carbohydrates exemplified by starch,glucose, lactose, sucrose, and dextrans, anti-oxidants exemplified byascorbic acid and glutathione, and hydrolyzed proteins). Exemplarydiluents include water, physiological saline solution, human serumalbumin, oils, polyethylene glycols, glycerine, propylene glycol, orother synthetic solvents. The compositions can also includeantibacterial agents such as benzyl alcohol, antioxidants such asascorbic acid or sodium bisulphite, chelating agents such as ethylenediamine-tetra-acetic acid, buffers such as acetates, citrates orphosphates, and agents for adjusting the osmolarity, such as sodiumchloride or dextrose.

In some embodiments, the immunogenic composition can be formulated foradministration by injection via the intramuscular, intraperitoneal,intradermal, or subcutaneous routes; or via mucosal administration tothe oral/alimentary, respiratory (e.g., intranasal administration),genitourinary tracts. Although the immunogenic composition can beadministered as a single dose, components thereof can also beco-administered together at the same time or at different times. Inaddition to a single route of administration, two or more differentroutes of administration can be used.

Immunogenic compositions can be lyophilized or be in aqueous form, e.g.,solutions or suspensions. Liquid formulations allow the compositions tobe administered directly from their packaged form, without the need forreconstitution in an aqueous medium. Compositions can be presented invials, or they can be presented in ready-filled syringes. The syringescan be supplied with or without needles. A syringe will include a singledose of the composition, whereas a vial can include a single dose ormultiple doses (e.g. 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses). In oneembodiment, the dose is for use in a human In a further embodiment, thedose is for an adult, adolescent, toddler, infant, or less than one yearold human, and can be administered by injection. Kits can include ameasured dose for administration to a subject.

An immunogenic composition can be lyophilized. When an immunogeniccomposition requires reconstitution, it can be provided in the form of akit which can comprise two vials, or can comprise one ready-filledsyringe and one vial, with the contents of the syringe being used toreconstitute the contents of the vial prior to injection.

The OMVs can be used in conjunction with other agents, such as anothervaccine or therapeutic agent. In some embodiments, the vaccine can be ameningococcal vaccine, such as a conjugate vaccine or a polysaccharidevaccine. In specific non-limiting examples, the vaccine is MPSV4(MENOMUNE®), MCV4 (MENACTRA®, MENHIBRIX®, MENVEO®) or a serogroup Bmeningococcal vaccine (TRUMENBA® and BEXSERO®). Additional vaccines areMENCEVAX®, a purified polysaccharide vaccine, such asNmVac4-A/C/Y/W-135, and NIMENTRIX®.

Methods are disclosed herein for inducing an immune response toNeisseria in a mammalian subject using any of the disclosed immunogeniccompositions including an effective amount of outer membranemicrovesicles (for example, OMV and/or blebs) from PorA⁻PorB⁻ N.meningitidis, such as a PorA⁻PorB⁻rmpM⁻ N. meningitidis. The immuneresponse can be a protective immune response or a therapeutic immuneresponse. The subject can be a human or veterinary subject. The subjectcan be an adult or a juvenile subject. In some embodiments, the subjectis a human of two weeks, one month, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11months, or one year or 15, 18, or 21 months of age, or a child of 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, or 13 years of age. The subject can be anadult, such as a human subject 18 or more years of age. The method caninclude administering 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 doses of aneffective amount of outer membrane microvesicles (for example, OMVand/or blebs) from PorA⁻PorB⁻ N. meningitidis, such as a PorA⁻PorB⁻rmpM⁻N. meningitidis. In some embodiments, a single dose is used. In otherembodiments, multiple doses are used, such as in a prime boost protocol.Exemplary non-limiting protocols are shown in the examples section. Aninitial dose and an additional dose can be administered within days,weeks, or months of each other.

In some embodiments, the subject has a N. meningitidis infection, andadministration of the immunogenic composition increases clearance of theN. meningitidis. The N. meningitidis in the immunogenic composition canbe any serogroup, such as serogroup A, B, or C. The N. meningitidisinfection can be of any serogroup, such as serogroup A, B, and C.Optionally, the N. meningitidis in the immunogenic composition and theN. meningitidis infection are the same serogroup.

In other embodiments, the subject has a N. gonorrhoeae infection, andadministration of the immunogenic composition increases clearance of theN. gonorrhoeae. In further embodiments, the subject is a healthysubject, and does not have a N. meningitidis or a N. gonorrhoeaeinfection. In some embodiments, methods are provided for inducing animmune response to N. gonorrhoeae in a mammalian subject, comprisingadministering to the mammalian subject an immunogenic compositioncomprising an effective amount of outer membrane microvesicles, such asblebs and/or OMVs, from PorA⁻PorB⁻ N. meningitidis and apharmaceutically acceptable carrier. The PorA⁻PorB⁻ N. meningitidis canbe RmpM⁻. The N. meningitidis can be serogroup A, B or C, or any otherserogroup (W, Y, etc.). The N. gonorrhoeae can be of any serotype.

The methods of the invention provide for administration of one or moreantigenic compositions to a mammalian subject (e.g., a human) to elicitan immune response. The immune response can be against more than onestrain of Neisseria species bacteria, and thus protection againstdisease caused by such bacteria. The disclosed methods can provide foran immunoprotective immune response against a 1, 2, 3, 4, 5 or morestrains of N. meningitidis species, where the strains differ in at leastone of serogroup, serotype, or serosubtype. See U.S. Published PatentApplication No. US 20170065699, incorporated herein by reference, whichdiscloses methods for immunizing against multiple strains, such as 2, 3,4, 5, or more strains.

Optionally, the immunogenic composition can include an adjuvant.Suitable adjuvants are disclosed above.

The immunogenic composition can be administered by any route. Thisincludes via injection for the intramuscular, intraperitoneal,intradermal, or subcutaneous routes; or via mucosal administration tothe oral/alimentary, respiratory (e.g., intranasal administration),genitourinary tracts. Although the immunogenic composition can beadministered as a single dose, additional doses can be co-administeredtogether at the same time or at different times. In addition to a singleroute of administration, two or more different routes of administrationcan be used.

Administration can be accomplished by single or multiple doses. The doseadministered to a subject in the context of the present disclosureshould be sufficient to induce a beneficial therapeutic response in asubject over time, or to inhibit or prevent infection. The dose requiredwill vary from subject to subject depending on the species, age, weight,and general condition of the subject, the severity of the infectionbeing treated, the particular composition being used, and its mode ofadministration. An appropriate dose can be determined by one of ordinaryskill in the art using only routine experimentation.

In some embodiments, an immunogenic composition can be administeredorally, nasally, nasopharyngeally, parenterally, enterically,gastrically, topically, transdermally, subcutaneously, intramuscularly,in tablet, solid, powdered, liquid, aerosol form, locally orsystemically, with or without added excipients. Actual methods forpreparing parenterally administrable compositions are described in suchpublications as Remington's Pharmaceutical Science, 15th ed., MackPublishing Company, Easton, Pa. (1980).

For oral administration, the compositions may need to be protected fromdigestion. This is typically accomplished either by association of thecomposition with an agent that renders it resistant to acidic andenzymatic hydrolysis or by packaging the composition in an appropriatelyresistant carrier. Means of protecting from digestion are well known inthe art.

The immunogenic compositions are administered to an animal that has oris at risk for acquiring a Neisseria infection, to prevent or at leastpartially arrest the development of disease and its complications. Anamount adequate to accomplish this is defined as an “effective dose” oran “immunogenically effective amount.” Amounts effective for use willdepend on, e.g., the antigenic composition, the manner ofadministration, the weight and general state of health of the subject,and the judgment of the prescribing physician. Single or multiple dosesof the antigenic compositions may be administered depending on thedosage and frequency required and tolerated by the patient, and route ofadministration. A prime boost strategy can be utilized.

The amount of outer membrane microvesicles included in the immunogeniccomposition is sufficient to elicit an immune response, such as ahumoral immune response and/or a cellular immune response, in thesubject. Amounts for the immunization of the mixture generally rangefrom about 0.001 mg to about 1.0 mg per 70 kilogram patient, morecommonly from about 0.001 mg to about 0.2 mg per 70 kilogram patient.Dosages from 0.001 up to about 10 mg per patient per day may be used,particularly when the antigen is administered to a secluded site and notinto the bloodstream, such as into a body cavity or into a lumen of anorgan. Substantially higher dosages (e.g. 10 to 100 mg or more) arepossible in oral, nasal, or topical administration. The initialadministration of the mixture can be followed by booster immunization ofthe same of different mixture, with at least one booster, such as twoboosters.

In some embodiments, administration is initiated prior to the first signof disease symptoms, or at the first sign of possible or actual exposureto pathogenic Neisseria. Without being bound by theory, immunoprotectiveantibodies for N. meningitidis and/or N. gonorrhoeae can be generated byimmunization with an immunogenic composition.

Immunoprotective antibodies for N. meningitidis and/or N. gonorrhoeaecan be administered to an individual (e.g., a human patient) to providefor passive immunity, either to prevent infection or disease fromoccurring, or as a therapy to improve the clinical outcome in patientswith established disease (e.g. decreased complication rate such asshock, decreased mortality rate, or decreased morbidity, such asdeafness). Antibodies administered to a subject that is of a speciesother than the species in which they are raised are often immunogenic.Thus, for example, murine or porcine antibodies administered to a humanoften induce an immunologic response against the antibody. Theimmunogenic properties of the antibody are reduced by altering portions,or all, of the antibody into characteristically human sequences therebyproducing chimeric or human antibodies, respectively.

Chimeric antibodies are immunoglobulin molecules comprising a human andnon-human portion. More specifically, the antigen combining region (orvariable region) of a humanized chimeric antibody is derived from anon-human source (e.g. murine), and the constant region of the chimericantibody (which confers biological effector function to theimmunoglobulin) is derived from a human source. The chimeric antibodyshould have the antigen binding specificity of the non-human antibodymolecule and the effector function conferred by the human antibodymolecule. A large number of methods of generating chimeric antibodiesare well known to those of skill in the art (see, e.g., U.S. Pat. Nos.5,502,167, 5,500,362, 5,491,088, 5,482,856, 5,472,693, 5,354,847,5,292,867, 5,231,026, 5,204,244, 5,202,238, 5,169,939, 5,081,235,5,075,431 and 4,975,369). An alternative approach is the generation ofhumanized antibodies by linking the CDR regions of non-human antibodiesto human constant regions by recombinant DNA techniques. See Queen etal., Proc. Natl. Acad. Sci. USA 86: 10029-10033 (1989) and WO 90/07861.

Fully human antibodies are also of use. Human antibodies consistentirely of characteristically human polypeptide sequences. The humanantibodies of this invention can be produced by a wide variety ofmethods (see, e.g., Larrick et al., U.S. Pat. No. 5,001,065). In oneembodiment, the human antibodies of the present invention are producedinitially in trioma cells (descended from three cells, two human and onemouse). Genes encoding the antibodies are then cloned and expressed inother cells, particularly non-human mammalian cells. The generalapproach for producing human antibodies by trioma technology has beendescribed by Ostberg et al. (1983), Hybridoma 2: 361-367, Ostberg, U.S.Pat. No. 4,634,664, and Engelman et al., U.S. Pat. No. 4,634,666.Triomas have been found to produce antibody more stably than ordinaryhybridomas made from human cells.

Methods for producing and formulating antibodies suitable foradministration to a subject (e.g., a human subject) are well known inthe art. For example, antibodies can be provided in a pharmaceuticalcomposition comprising an effective amount of an antibody and apharmaceutical excipient (e.g., saline). The pharmaceutical compositionmay optionally include other additives (e.g., buffers, stabilizers,preservatives, and the like). An effective amount of antibody isgenerally an amount effective to provide for protection againstNeisserial disease or symptoms for a desired period, e.g., a period ofat least about 2 days to 10 days or 1 month to 2 months).

Chimeric Recombinant PorB

Chimeric recombinant PorB polypeptides are shown in FIGS. 11A-11B andSEQ ID NOs; 39-51. These PorB sequences are not naturally occurring.These chimeric recombinant PorB polypeptide can be included in aNeisseria. A Neisseria strain expressing the chimeric recombinant PorBis PorB⁺. Polypeptides can be produced that are at least 95%, 96%, 97%,98% or 99% identical to these proteins.

Nucleic acid molecules, such as DNA and RNA, can be produced encodingthese chimeric recombinant PorB polypeptides. These polynucleotidesinclude DNA, cDNA, and RNA sequences which encode the polypeptide ofinterest. Silent mutations in the coding sequence result from thedegeneracy (i.e., redundancy) of the genetic code, whereby more than onecodon can encode the same amino acid residue. Thus, for example, leucinecan be encoded by CTT, CTC, CTA, CTG, TTA, or TTG; serine can be encodedby TCT, TCC, TCA, TCG, AGT, or AGC; asparagine can be encoded by AAT orAAC; aspartic acid can be encoded by GAT or GAC; cysteine can be encodedby TGT or TGC; alanine can be encoded by GCT, GCC, GCA, or GCG;glutamine can be encoded by CAA or CAG; tyrosine can be encoded by TATor TAC; and isoleucine can be encoded by ATT, ATC, or ATA. Tablesshowing the standard genetic code can be found in various sources (e.g.,L. Stryer, 1988, Biochemistry, 3.sup.rd Edition, W.H. 5 Freeman and Co.,NY). Nucleic acid molecules can readily be produced by one of skill inthe art, using the amino acid sequences provided herein, and the geneticcode.

A nucleic acid molecule can be cloned or amplified by in vitro methods,such as the polymerase chain reaction (PCR), the ligase chain reaction(LCR), the transcription-based amplification system (TAS), theself-sustained sequence replication system (3SR) and the Qβ replicaseamplification system (QB). For example, a polynucleotide encoding theprotein can be isolated by polymerase chain reaction of cDNA usingprimers based on the DNA sequence of the molecule. A wide variety ofcloning and in vitro amplification methodologies are well known topersons skilled in the art. PCR methods are described in, for example,U.S. Pat. No. 4,683,195; Mullis et al., Cold Spring Harbor Symp. Quant.Biol. 51:263, 1987; and Erlich, ed., PCR Technology, (Stockton Press,NY, 1989).

A nucleic acid sequence that encodes a chimeric recombinant PorBpolypeptide can be incorporated into a vector capable of expression in ahost cell, using established molecular biology procedures. For examplenucleic acids, such as cDNAs, that encode the chimeric recombinant PorBpolypeptide can be manipulated with standard procedures such asrestriction enzyme digestion, fill-in with DNA polymerase, deletion byexonuclease, extension by terminal deoxynucleotide transferase, ligationof synthetic or cloned DNA sequences, site-directed sequence-alterationvia single-stranded bacteriophage intermediate or with the use ofspecific oligonucleotides in combination with PCR or other in vitroamplification.

Exemplary procedures sufficient to guide one of ordinary skill in theart through the production of vector capable of expression in a hostcell (such as an adenoviral vector) that includes a polynucleotidesequence that encodes a chimeric recombinant PorB polypeptide can befound for example in Sambrook et al., Molecular Cloning: A LaboratoryManual, 2d ed., Cold Spring Harbor Laboratory Press, 1989; Sambrook etal., Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring HarborPress, 2001; Ausubel et al., Current Protocols in Molecular Biology,Greene Publishing Associates, 1992 (and Supplements to 2003); andAusubel et al., Short Protocols in Molecular Biology: A Compendium ofMethods from Current Protocols in Molecular Biology, 4th ed., Wiley &Sons, 1999.

Typically, a polynucleotide sequence encoding a chimeric recombinantPorB polypeptide is operably linked to transcriptional control sequencesincluding, for example a promoter. A promoter is a polynucleotidesequence recognized by the transcriptional machinery of the host cell(or introduced synthetic machinery) that is involved in the initiationof transcription.

Exemplary promoters include viral promoters, such as cytomegalovirusimmediate early gene promoter (“CMV”), herpes simplex virus thymidinekinase (“tk”), SV40 early transcription unit, polyoma, retroviruses,papilloma virus, hepatitis B virus, and human and simianimmunodeficiency viruses. Other promoters are isolated from mammaliangenes, including the immunoglobulin heavy chain, immunoglobulin lightchain, T-cell receptor, HLA DQ α and DQ β, β-interferon, interleukin-2,interleukin-2 receptor, MHC class II, HLA-DRα, β-actin, muscle creatinekinase, prealbumin (transthyretin), elastase I, metallothionein,collagenase, albumin, fetoprotein, β-globin, c-fos, c-HA-ras, insulin,neural cell adhesion molecule (NCAM), α1-antitrypsin, H2B (TH2B)histone, type I collagen, glucose-regulated proteins (GRP94 and GRP78),rat growth hormone, human serum amyloid A (SAA), troponin I (TNI),platelet-derived growth factor, and dystrophin, and promoters specificfor keratinocytes, and epithelial cells.

The promoter can be either inducible or constitutive. An induciblepromoter is a promoter which is inactive or exhibits low activity exceptin the presence of an inducer substance. Examples of inducible promotersinclude, but are not limited to, MT II, MMTV, collagenase, stromelysin,SV40, murine MX gene, α-2-macroglobulin, MHC class I gene h-2kb, HSP70,proliferin, tumor necrosis factor, or thyroid stimulating hormone genepromoter.

Typically, the promoter is a constitutive promoter that results in highlevels of transcription upon introduction into a host cell in theabsence of additional factors. Optionally, the transcription controlsequences include one or more enhancer elements, which are bindingrecognition sites for one or more transcription factors that increasetranscription above that observed for the minimal promoter alone.

It may be desirable to include a polyadenylation signal to effect propertermination and polyadenylation of the gene transcript. Exemplarypolyadenylation signals have been isolated from bovine growth hormone,SV40 and the herpes simplex virus thymidine kinase genes. Any of theseor other polyadenylation signals can be utilized in the context of theadenovirus vectors described herein.

The polynucleotides encoding a chimeric recombinant PorB polypeptideinclude a recombinant DNA which is incorporated into a vector in anautonomously replicating plasmid or virus or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (such asa cDNA) independent of other sequences. The nucleotides can beribonucleotides, deoxyribonucleotides, or modified forms of eithernucleotide. The term includes single and double forms of DNA.

Viral vectors can also be prepared encoding the chimeric recombinantPorB polypeptide. A number of viral vectors have been constructed,including polyoma, SV40 (Madzak et al., 1992, J. Gen. Virol.,73:15331536), adenovirus (Berkner, 1992, Cur. Top. Microbiol. Immunol.,158:39-6; Berliner et al., 1988, Bio Techniques, 6:616-629; Gorziglia etal., 1992, J. Virol., 66:4407-4412; Quantin et al., 1992, Proc. Nad.Acad. Sci. USA, 89:2581-2584; Rosenfeld et al., 1992, Cell, 68:143-155;Wilkinson et al., 1992, Nucl. Acids Res., 20:2233-2239;Stratford-Perricaudet et al., 1990, Hum. Gene Ther., 1:241-256),vaccinia virus (Mackett et al., 1992, Biotechnology, 24:495-499),adeno-associated virus (Muzyczka, 1992, Curr. Top. Microbiol. Immunol.,158:91-123; On et al., 1990, Gene, 89:279-282), herpes viruses includingHSV and EBV (Margolskee, 1992, Curr. Top. Microbiol. Immunol.,158:67-90; Johnson et al., 1992, J. Virol., 66:29522965; Fink et al.,1992, Hum. Gene Ther. 3:11-19; Breakfield et al., 1987, Mol. Neurobiol.,1:337-371; Fresse et al., 1990, Biochem. Pharmacol., 40:2189-2199),Sindbis viruses (H. Herweijer et al., 1995, Human Gene Therapy6:1161-1167; U.S. Pat. Nos. 5,091,309 and 5,2217,879), alphaviruses (S.Schlesinger, 1993, Trends Biotechnol. 11:18-22; I. Frolov et al., 1996,Proc. Natl. Acad. Sci. USA 93:11371-11377) and retroviruses of avian(Brandyopadhyay et al., 1984, Mol. Cell Biol., 4:749-754; Petropouploset al., 1992, J. Virol., 66:3391-3397), murine (Miller, 1992, Curr. Top.Microbiol. Immunol., 158:1-24; Miller et al., 1985, Mol. Cell Biol.,5:431-437; Sorge et al., 1984, Mol. Cell Biol., 4:1730-1737; Mann etal., 1985, J. Virol., 54:401-407), and human origin (Page et al., 1990,J. Virol., 64:5370-5276; Buchschalcher et al., 1992, J. Virol.,66:2731-2739). Baculovirus (Autographa californica multinuclearpolyhedrosis virus; AcMNPV) vectors are also known in the art, and maybe obtained from commercial sources (such as PharMingen, San Diego,Calif.; Protein Sciences Corp., Meriden, Conn.; Stratagene, La Jolla,Calif.). Thus, in one embodiment, the polynucleotide encoding a chimericrecombinant PorB polypeptide is included in a viral vector. Suitablevectors include retrovirus vectors, orthopox vectors, avipox vectors,fowlpox vectors, capripox vectors, suipox vectors, adenoviral vectors,herpes virus vectors, alpha virus vectors, baculovirus vectors, Sindbisvirus vectors, vaccinia virus vectors and poliovirus vectors. Specificexemplary vectors are poxvirus vectors such as vaccinia virus, fowlpoxvirus and a highly attenuated vaccinia virus (MVA), adenovirus,baculovirus, yeast and the like.

The chimeric recombinant PorB polypeptide, or a polynucleotide encodingthe chimeric recombinant PorB polypeptide, as disclosed herein can beformulated in a variety of ways. Pharmaceutical compositions can includethe chimeric recombinant PorB polypeptide, and pharmaceuticallyacceptable carrier, and optionally an adjuvant. Pharmaceuticallyacceptable carriers and adjuvants are disclosed above and are of usewith a disclosed chimeric recombinant PorB polypeptide, or apolynucleotide encoding the chimeric recombinant PorB polypeptide.

EXAMPLES

The porin protein PorB is the dominant outer membrane protein (OMP) onthe meningococcal surface and has been proposed as a MenB vaccinecandidate. Expressed in all known clinical isolates, it is believed tobe essential for in vivo meningococcal survival and is not subject tophase variation (Abad et al. (2006) Clin Vaccine Immunol 13: 1087-1091).Both native and recombinant PorB elicit the production of bactericidalantibodies (Poolman et al. (1983) Infect Immun 40: 398-406, Wright etal. (2002) Infect Immun 70: 4028-4034), and PorB-specific antibodies aregenerated in response to immunization with N. meningitidis outermembrane vesicle (OMV) vaccines (Bash et al. (2000) FEMS Immunol MedMicrobiol 29: 169-176, Marzoa et al. (2012) Vaccine 30: 2387-2395,Norheim et al. (2012) Scand J. Immunol 76: 99-107), highlighting thedirect interaction of PorB with the host immune system.

PorB-induced antibody responses are directed against six variablesurface-exposed loops that differ in sequence depending on serotype.Although N. meningitidis is naturally competent and porB geneticmosaicism provides evidence for horizontal genetic exchange and strongpositive selection, the sequences of some PorB serotypes commonlyassociated with invasive disease are often conserved, calling intoquestion the interaction of specific PorB loop sequences in immuneengagement.

Example 1 Chimeric Recombinant PorB (rPorB) Proteins and IsogenicPorB-Expressing Strains

In this example, it was demonstrated that antibody binding to a PorBepitope can be altered by sequence mutations in non-epitope loops.Through the construction of chimeric PorB types (see FIGS. 11A-11B) andPorB molecular dynamics simulations, it was demonstrated that loops bothadjacent and non-adjacent to the epitope loop can enhance or diminishantibody binding, a phenotype that correlates with serum bactericidalactivity.

PorB can self-assemble into homotrimers and is known to interact withother OMPs to form larger protein complexes. A panel of selective OMPdeletion mutant strains (FIG. 1 ) was engineered to analyze the impactof complex formation on PorB-specific antibody binding (FIG. 2 ).Deletion of PorA and RmpM resulted in decreased antibody binding to PorBand lower bactericidal titers in a PorB-dependent killing assay.

Example 2 Serum Bactericidal Assays

Z15 is a bactericidal mAb; serum bactericidal assays (SBAs) wereperformed to assess complement-mediated killing. Although titers werelow, the only strains in the isogenic hybrid strains expressing chimericPorB that were consistently killed were those that were bound by Z15most efficiently in whole cell dot blot assays, namely the MC58PorB-expressing strain and the two L54-L64 mutants, M(1-4)C(5-6)M(7-8)and M(1-4)B(5-6)M(7-8). Thus, decreases in mAb binding associated withchanges to non-epitope sequences correlated with a reduction inantibody-dependent killing activity.

Example 3 OMV from PorA(−) Isogenic Strains with Various PorB

In the following tables, the immunizing antigen used to generate theimmune sera is listed in the far left column and the strain that isbeing tested (killed) in the bactericidal assay is shown in the firstrow at the top of each column. The numbers are the reciprocal of thehighest dilution that kills at least 50% of the bacteria. Hence, ahigher number means that there is more potent killing i.e. a higherconcentration of functional (protective) antibodies.

Table A below is from the experiments with OMV from PorA(−) isogenicstrains (MC58 background) but with different PorB. The antiseragenerated killed all 4 wild type strains tested. The high titers againstthe MC58 strain for all can be explained because that strain ishomologous to the parental strain from which the OMV were derived,except for expression of a heterologous PorB type and the lack of PorAexpression. Overall, the greatest cross-protection is that induced bythe OCh OMV (FIGS. 11A-11B) immunogen in which the PorB is a constructedchimeric PorB not found in nature that was not immunogenic.

TABLE A MC58ΔporA::porB MC58 Cu385 Ch501 BB1350 OMV WT WT WT WT Mc58PorB Rabbit 1 64 8 32 128 Mc58 PorB Rabbit 2 512 4 32 16 Bb1350 PorBRabbit 1 256 16 16 16 Bb1350 PorB Rabbit 2 256 16 16 512 Ch501 PorBRabbit 1 512 16 32 128 Ch501PorB Rabbit 2 512 8 64 16 Och PorB Rabbit 1512 64 128 256 Och PorB Rabbit 2 512 64 256 256 Cu385 rabbit 1 1024 12864 16 Cu385 Rabbit 2 512 128 64 16Table B is from Bash et al. FEMS Immunol Med Microbiol. 2000 November;29(3):169-76) wherein the OMV are from wild type strains and theantisera tested against the same wild type strains (nomenclaturefollowing strain in top column shows serogroup (capsule); serotype(PorB); subserotype (PorA). The M1.2 strain is PorA and class 5negative. Importantly H355 is a similar to MC58 and has the same PorA asCu385. In this experiment, the Cu385 antisera has lowered bactericidalactivity, but the H355 antisera is bactericidal—but only against the twostrains sharing the same type of PorA. Table B illustrates the PorAspecificity of immune responses to OMV containing PorA.

TABLE B Ch501 Cu385 M1.2 H355 OMV (B:4, 15; nss) (B:4:P1.15)(B:4:P1-:P5-) (B:15:P1.15) Ch501 Rabbit 1  6 to 12 12 to 48  6 to 12 6to 12 Ch501 Rabbit 2 12 to 48 12 to 48 12 to 48 6 to 12 Ch501 Rabbit 312 to 48 12 to 48 12 to 48 — Cu385 Rabbit 1 — — — — Cu385 Rabbit 2 —  6to 12 — — H355 Rabbit 1 — >48 — >48 H355 Rabbit 2 — >48 — >48

Example 7 Single, Double, or Triple Deletions of the Genes Encoding theOuter Membrane Proteins PorA (ΔA), PorB (ΔB), and the OMP stabilizingprotein RmpM (ΔR)

In this example, mutants were generated with single, double, or tripledeletions of the genes encoding PorA (PorA⁻, or ΔA), PorB (PorB⁻, orΔB), and the OMP stabilizing protein RmpM (RmpM⁻, or ΔR) in the MC58background.

Development and Characterization: Because PorB is most often found inthe context of a homotrimer or in association with PorA in heterotrimers(Sanchez et al. (2009) Vaccine 27: 5338-5343, Marzoa et al. (2009)Proteomics 9: 648-656), the possibility was explored that differences inthe expression of PorB-interacting OMPs can alter the binding ofPorB-specific antibodies. A series of mutants were generated withsingle, double, or triple deletions of the genes encoding PorA (ΔA),PorB (ΔB), and RmpM (ΔR) in the MC58 background. Proper deletion wasconfirmed by dot blot, PCR, and sequencing analyses (FIG. 1A). OMVsobtained from each of the strains exhibited no difference in size orpurity when assessed by OptiPrep density centrifugation and transmissionelectron microscopy, though both the ΔAB and ΔABR OMVs and theirrespective strains expressed approximately ˜2-fold less capsule relativeto the other OMVs/strains when probed with anti-capsular SEAM12 in a dotblot. When analyzed via SDS-PAGE (FIG. 1B), there were no majordifferences in banding patterns observed, except for the expected lossof expression of PorA and PorB in the respective deletion strains andelevated PorA levels in ΔB and ΔBR, a phenomenon that has beenpreviously described for the porB single mutant (Peak et al., 2015).Accordingly, ΔB exhibited an increase in porA transcript levels (foldchange of 2.83±0.09 relative to WT) when examined by quantitative realtime PCR (qPCR), though porB levels in ΔA were unaltered. Despitesimilar PorB and capsule expression, all of the PorA- and RmpM-deficientOMVs exhibited diminished binding profiles relative to the wild type(WT) when used to competitively inhibit Z15 binding to rMC58 (FIG. 2A,left panel), a phenotype consistent with lower bactericidal titers in aZ15-dependent SBA (Table 1). Binding of P7 to PorA was only decreased inΔR compared to the other strains (FIG. 2B, left panel).

TABLE 1 Z15 PorB monoclonal antibody-dependent bactericidal activity ofhuman sera against MC58 WT strain and isogenic OMP single, double, andtriple deletion mutants. Titers are depicted as the reciprocal of thehighest dilution resulting in ≥ 50% killing relative to heat-inactivatedcontrol wells lacking Z15. Results shown were the same for twoindependent assays Strain Titer WT >1024 ΔA 32 ΔB <4 ΔR 256 ΔAB <4 ΔAR128 ΔBR <4 ΔABR <4

When the OMVs were fractionated by blue native gel electrophoresis(BNGE) and probed with the same antibodies to examine OMP complexformation, differences in banding patterns emerged. Whereas ΔA exhibiteda phenotype similar to the WT after probing with Z15 (FIG. 2A, rightpanel) and ΔB adsorbed polyclonal sera MB1 and MB2, ΔR and ΔAR werecharacterized by only one large band that was of lower molecular weight(MW). This same band in ΔR also tested positive for the presence of PorA(FIG. 2B, right panel). When porB was deleted singly or in combinationwith rmpM, probing with P7 demonstrated the presence of one band thatwas roughly of the same MW found in the WT and three that were of higherMW (FIG. 2B, right panel). The largest of these (˜700 kDa) has beenconfirmed previously as a high MW complex consisting of PorA by massspectrometry (Marzoa et al. (2009) Proteomics 9: 648-656).

porA, porB, and rmpM gene knockouts: The differences observed viaimmunoblot suggested that OMP deletion led to formation of atypicalPorA- and PorB-containing complexes. To assess their composition, BNGEwas performed on OMVs. Post-staining with COOMASSIE® R-250 revealed thesame dominant bands observed in the western blot (FIG. 2C, upper panel),though multiple higher and lower MW bands were visible by silver stain(FIG. 2D, lower panel). The dominant bands were cut out and in-geldigestion performed, followed by mass spectrometry for identification.Analysis of the recovered peptides revealed the absence of PorA, PorB,and RmpM in the complexes of the respective knockout strains and thepresence of multiple proteins that were not detected in the WTcomplexes, including those involved in protein refolding (GroEL andDsbD), amino acid synthesis (IlvC), glycolysis (GapA, AceE), andelectron transport chain-dependent metabolism (FixN, NqrA) (Table 2). Inaddition to expressing many of these same proteins, the probableTonB-dependent receptor TdfH was also found in the dominant complexes ofthe ΔBR and ΔABR strains, suggesting that deletion of PorB and RmpM intandem diminishes nutrient acquisition and can lead to alterations inmeningococcal gene expression and metabolism. Viability assays were usedto support this study, which showed decreased growth for the strainsrelative to the WT over a time course of 8 h (FIG. 3A), including asignificant reduction in the number of colony forming units (CFUs) (FIG.3B) and production of smaller colonies as assessed by growth on BHIsolid medium (FIG. 3C). While the ΔB and the ΔAB mutants were alsocharacterized by a small colony phenotype (FIG. 3C), this effect washeterogeneous and maintained over repeated culturing, suggesting thepresence of a non-PorA-specific protein that could compensate for smallcolony morphology in the presence of RmpM.

TABLE 2 List of proteins identified from dominant complexes of OMPdeletion mutant OMVs. OMVs were fractionated via blue native gelelectrophoresis, visualized with COOMASSIE ® staining, and cut out fromthe gel. Following SDS removal and trypsinization, peptides wereextracted from the gel fragments and identified using reverse phaseone-dimensional liquid chromatography mass spectrometry (1D LC-MS/MS).The complex from which each protein is derived is noted and correspondsto the bands observed in FIG. 2C. Proteins listed in bold indicate thosenot identified in WT complexes. Uniprot Accession numbers are providedand are incorporated by reference as available on Jul. 21, 2017. StrainComponents Function UnitProt ID Complex WT PorB Class 3 OMP; porinQ6LD38 1, 2 PorA Class 1 OMP; porin P0DH58 1, 2 RmpM Class 4 OMP;membrane integrity protein P0A0V3 1, 2 Opa Opacity protein O30755 1, 2Opc Class 5 OMP; adhesin Q9AE79 1, 2 NMB0378 Inorganic phosphatetransporter Q7DDQ8 1, 2 MtrE Multidrug efflux pump channel proteinQ9JY68 1 TbpA Transferrin-binding protein Q9JPJ0 1, 2 NMA1697 Probablelipoprotein A0A0H5QU 1, 2 LpdA2 Dihydrolipoyl dehydrogenase W8 1, 2 LptDLPS-assembly protein Q9JZ09 1, 2 NMB1964 Uncharacterized protein Q9K1871 AniA Copper-containing nitrite reductase Q7DD60 1, 2 NorB Nitric oxidereductase Q9JYE1 1 Pnp Polyribonucleotide nucleotidyltransferase Q9JYE21, 2 PetB Cytochrome b Q9K062 2 PetC Ubiquinol--cytochrome c reductase,cytochrome Q9JXH0 2 NMB1805 c1 Q9JXH1 2 NMB1677 Cytochrome c4 Q7DD79 1FixO Cytochrome c5 Q9JYA2 1, 2 FixP Cytochrome c oxidase subunit IIQ9JY59 2 Cytochrome c oxidase subunit III E1AA24 ΔA PorB Class 3 OMP;porin Q6LD38 3, 4 RmpM Class 4 OMP; membrane integrity protein P0A0V3 3,4 Opa Opacity protein O30755 3, 4 Opc Class 5 OMP; adhesin Q9AE79 3, 4TbpA Transferrin-binding protein Q9JPJ0 3, 4 NMA1697 Probablelipoprotein A0A0H5QU 4 LpdA2 Dihydrolipoyl dehydrogenase W8 3, 4 LptDLPS-assembly protein Q9JZ09 4 AniA Copper-containing nitrite reductaseQ9K187 3, 4 NorB Nitric oxide reductase Q9JYE1 4 Pnp Polyribonucleotidenucleotidyltransferase Q9JYE2 3, 4 PetC Ubiquinol--cytochrome creductase, cytochrome Q9K062 3, 4 NMB1805 c1 Q9JXH1 3, 4 FixO Cytochromec4 Q7DD79 3, 4 FixP Cytochrome c oxidase subunit II Q9JY59 3, 4 GroELCytochrome c oxidase subunit III E1AA24 3 GapA 60 kDa chaperonin X5EPF83 DsbD Glyceraldehyde-3-phosphate dehydrogenase C6SEX6 4 Thiol:disulfideinterchange protein Q9JYM0 ΔB PorA Class 1 OMP; porin P0DH58 5, 6 RmpMClass 4 OMP; membrane integrity protein P0A0V3 5 Opa Opacity proteinO30755 5, 6 NMB0378 Inorganic phosphate transporter Q7DDQ8 6 MtrEMultidrug efflux pump channel protein Q9JY68 5 TbpA Transferrin-bindingprotein Q9JPJ0 5 LpdA2 Dihydrolipoyl dehydrogenase Q9JZ09 5 LptDLPS-assembly protein Q9K187 5, 6 NMB1964 Uncharacterized protein Q7DD605 AniA Copper-containing nitrite reductase Q9JYE1 5, 6 PnpPolyribonucleotide nucleotidyltransferase Q9K062 5, 6 PetCUbiquinol--cytochrome c reductase, cytochrome Q9JXH1 6 NMB1805 c1 Q7DD796 FixN Cytochrome c4 Q7DD90 6 FixO Cytochrome c oxidase subunit I Q9JY595, 6 DsbD Cytochrome c oxidase subunit II Q9JYM0 5 Thiol:disulfideinterchange protein ΔR PorB Class 3 OMP; porin Q6LD38 7 PorA Class 1OMP; porin P0DH58 7 Opa Opacity protein O30755 7 Opc Class 5 OMP;adhesin Q9AE79 7 PilE Fimbrial (pilin) protein P05431 7 TbpATransferrin-binding protein Q9JPJ0 7 NMA1697 Probable lipoproteinA0A0H5QU 7 LptD LPS-assembly protein W8 7 AniA Copper-containing nitritereductase Q9K187 7 AceE Pyruvate dehydrogenase subunit E1 Q9JYE1 7 SucBDihydrolipoyllysine-residue Q9JZ12 7 succinyltransferase component of 2-Q9JZP6 NMB1677 oxoglutarate dehydrogenase complex 7 FixN Cytochrome c5Q9JYA2 7 FixO Cytochrome c oxidase subunit I Q7DD90 7 FixP Cytochrome coxidase subunit II Q9JY59 7 NqrA Cytochrome c oxidase subunit III E1AA247 GroEL Na(+)-translocating NADH-quinone reductase Q9K0M3 7 Fhs subunitA X5EPF8 7 60 kDa chaperonin Q9JXY2 Formate--tetrahydrofolate ligase ΔABOpa Opacity protein O30755 8 NMB0378 Inorganic phosphate transporterQ7DDQ8 8 TbpA Transferrin-binding protein Q9JPJ0 8 AniACopper-containing nitrite reductase Q9JYE1 8 PetC Ubiquinol--cytochromec reductase, cytochrome Q9JXH1 8 NMB1805 c1 Q7DD79 8 FixN Cytochrome c4Q7DD90 8 FixO Cytochrome c oxidase subunit I Q9JY59 8 Cytochrome coxidase subunit II ΔAR TbpA Transferrin-binding protein Q9JPJ0 9 AniACopper-containing nitrite reductase Q9JYE1 9 FixO Cytochrome c oxidasesubunit II Q9JY59 9 ΔBR Por A Class 1 OMP; porin P0DH58 10 Opa Opacityprotein O30755 10, 11 NMB0378 Inorganic phosphate transporter Q7DDQ8 11MtrE Multidrug efflux pump channel protein Q9JY68 10 NMA1697 Probablelipoprotein A0A0H5QU 11 LpdA2 Dihydrolipoyl dehydrogenase W8 10, 11 LptDLPS-assembly protein Q9JZ09 11 TdfH Probable TonB-dependent receptorQ9K187 11 AniA Copper-containing nitrite reductase Q7DDB6 10, 11 PnpPolyribonucleotide nucleotidyltransferase Q9JYE1 10, 11 AceE Pyruvatedehydrogenase subunit E1 Q9K062 11 PetB Cytochrome b Q9JZ12 11 PetCUbiquinol--cytochrome c reductase, cytochrome Q9JXH0 11 NMB1805 c1Q9JXH1 11 FixN Cytochrome c4 Q7DD79 11 FixO Cytochrome c oxidase subunitI Q7DD90 11 FixP Cytochrome c oxidase subunit II Q9JY59 11 GroELCytochrome c oxidase subunit III E1AA24 11 GapA 60 kDa chaperonin X5EPF811 DsbD Glyceraldehyde-3-phosphate dehydrogenase C6SEX6 11 FhsThiol:disulfide interchange protein Q9JYM0 11 Formate--tetrahydrofolateligase Q9JXY2 ΔABR Opa Opacity protein O30755 12 NMB0378 Inorganicphosphate transporter Q7DDQ8 12 TbpA Transferrin-binding protein Q9JPJ012 LptD LPS-assembly protein Q9K187 12 TdfH Probable TonB-dependentreceptor Q7DDB6 12 AniA Copper-containing nitrite reductase Q9JYE1 12PetC Ubiquinol--cytochrome c reductase, cytochrome Q9JXH1 12 NMB1805 c1Q7DD79 12 FixN Cytochrome c4 Q7DD90 12 FixO Cytochrome c oxidase subunitI Q9JY59 12 IlvC Cytochrome c oxidase subunit II Q9JYI2 12 Ketol-acidreductoisomerase

TABLE 3Oligonucleotides used in this study. Restriction endonuclease sites and DNA uptakesequence (DUS) are depicted as underlined and in bold, respectively.*Primer residues numbered relative to MC58 sequence. SEQ ID Primer NameSequence (5’→3’) NO: DescriptionStrain construction: Immunizing OMV PorB antigens porBNdeIFGACTCATATGTTCAGACATGGAATCGCC  1 Forward primer, amplifiesfull porB ORF; contains NdeI restriction site. porBBamHIR ATATGGATCCTTCAGACGGCGCATTTTTATG  2 Reverse primer, amplifiesfull porB ORF; contains BamHI restriction site and DUS.Strain construction: OMP deletion mutants porBUPBamHIFATATGGATCCTGCAATGCCCTCCAATAC  3 Forward primer, amplifies~290 bp 5’ MC58 porB upstream region; contains BamHI restriction site.porBUPXbaIR CGCGTCTAGATGCTGTATTCCTTTTTTGGTTAA  4Reverse primer, amplifies ~290 bp 5’ MC58 porB upstream region; containsXbaI restriction site. porBDOWNSphIF ATATGCATGCTCTGCAAAGATTGGTATCAACA  5Forward primer, amplifies 310 bp 3’ MC58 porB downstream region;contains SphI restriction site. porBDOWNHindIIIR ATATAAGCTTCAGACGGCTGAAACTCAACG  6 Reverse primer, amplifies 310 bp 3’ MC58 porBdownstream region; contains Hindlll restriction site and DUS. eryXbaIFATATTCTAGACACCATAGGCTTTAGAGAAGTA  7 Forward primer, amplifies TTTGAATGC1.1 kb ermB cassette; contains XbaI restriction site. erySphIRCCGAGCATGCTTATTATTATTTCCTCCCGTTAA  8 Reverse primer, amplifies ATAATAG1.1 kb ermB cassette; contains SphI restriction site. porAUPBamHIFATATGGATCCAAGCCGAGACTGCATC  9 Forward primer, amplifies~260 bp 5’ MC58 porA upstream region; contains BamHI restriction site.porAUPXbaIR CGCGTCTAGAATCGGCTTCCTTTTGTAAAT 10 Reverse primer, amplifies~260 bp 5’ MC58 porA upstream region; contains XbaI restriction site.porADOWNSphIF ATATGCATGCATATCGGGGCGG 11 Forward primer, amplifies~250 bp 3’ MC58 porA downstream region; contains SphI restriction site.porADOWNHindIIIR ATATAAGCTT CAGACGGCGCATTTTTATGC 12Reverse primer, amplifies ~250 bp 3’ MC58 porA downstream region;contains HindIII restriction site and DUS. kanXbaIFATATTCTAGAGGGAAAGCCACTTTGTGTCTCA 13 Forward primer, amplifies~950 bp aph3A cassette; contains XbaI restriction site. kanSphIRGTATGCATGCTTATTATTAGAAAAACTCATCG 14 Reverse primer, amplifies AGCATC~950 bp aph3A cassette; contains SphI restriction site. rmpMUPBamHIFTAGTGGATCCAATCGTGCGATATGGAA 15 Forward primer, amplifies250 bp 5’ MC58 rmpM upstream region; contains BamHI restriction site.rmpMUPXbaIR CGCGTCTAGATTTATTCCCTCATTAAATTTGTA 16Reverse primer, amplifies CAGC ~250 bp 5’ MC58 rmpMupstream region; contains XbaI restriction site. rmpMDOWNSphIFGTATGCATGCGGCTAGGCAATATCTTG 17 Forward primer, amplifies~260 bp 3’ MC58 rmpM downstream region; contains SphI restriction site.rmpMDOWNHindIIIR ATATAAGCTT CAGACGGCGTTAATCCACTAT 18Reverse primer, amplifies AAAGC ~260 bp 3’ MC58 rmpM downstream region;contains HindIII restriction site and DUS. chlXbaIFGTATTCTAGACGCCGAATAAATACCTGTGACG 19 Forward primer, amplifies G~870 bp cat cassette; contains XbaI restriction site. chlSphIRATATGCATGCTTATTATTACGCCCCGCC 20 Reverse primer, amplifies~870 bp cat cassette; contains SphI restriction site.Strain construction: Hybrid PorB types* porBORFXbaIFATATTCTAGAATGAAAAAATCCCTGATTGCCC 21 Forward primer, porB TGACamino acid position Ml; contains XbalIrestriction site. porBF189RGAAGAAGCCACCGTTTTTGTAGTTGAAGC 22 Reverse primer, porBamino acid position F189; contains 15 bp overlapping with porBN185F.porBN185F AACGGTGGCTTCTTCGTGCAATATG 23 Forward primer, porBamino acid position N185; contains 15 bp overlapping with porBF189R.porBS267R AGAAACTCGGGGCGTTACGTTG 24 Reverse primer, porBamino acid position S267; contains 15 bp overlapping with porBT263F.porBT263F ACGCCCCGAGTTTCTTACGC25 25 Forward primer, porBamino acid position T263; contains 15 bp overlapping with porBS267R.porBORFSphIR ATATGCATGCTTAGAATTTGTGGCGCAG 26 Reverse primer, porBamino acid position F331; contains SphI restriction site. kanSphIFATATGCATGCGAAAGCCACTTTGTGTCT 27 Forward primer, usedwith kanSphIR to amplify ~950 bp aph3A cassette;contains SphI restriction site. rPorB/rPorA synthesis porBNdeIF2ATATCATATGGACGTTACCCTGTACGGCA 28 Forward primer, amplifiesporB ORF starting at amino acid D20; contains Ndel restriction site.porBBlpIR ATATGCTCAGCTTAGAATTTGTGGCGC 29 Reverse primer, amplifiesporB ORF starting at amino acid D20; contains BlpI restriction site.porANdelF ATATCATATGGATGTCAGCCTATACGGCGA 30 Forward primer, amplifiesporA ORF starting at amino acid D20; contains NdeI restriction site.porABlpIR ATATCTCGAGGAATTTGTGGCGCAAAC 31 Forward primer, amplifiesporA ORF starting at amino acid D20; contains BlpI restriction site.qPCR analysis porAF TGTCGGACGTAATGCTTTTG 32 Forward primer, amplifies199 bp porA transcript. porAR GGCAATTTCGGTCGTACTGT 33Reverse primer, amplifies 199 bp porA transcript. porBFCAATACGCGCTTAACGACAA 34 Forward primer, amplifies200 bp porB transcript. porBR GAAGCGTACAGGGCATCATT 35Reverse primer, amplifies 200 bp porB transcript. 16SFGCGCAACCCTTGTCATTAGT 36 Forward primer, amplifies 198 bp 16S rRNAtranscript. 16SR CGGACTACGATCGGTTTTGT 37 Reverse primer, amplifies198 bp 16S rRNA transcript.

Example 8 Materials and Methods for Examples 1-7

Bacterial growth conditions: N. meningitidis strains were routinelyincubated overnight at 37 degrees Celsius in the presence of 5 percentCO₂ on BBL Brain Heart Infusion (BHI) Agar plates (Becton Dickinson,Sparks, Md.) supplemented with heat inactivated 5 percent HyClone DonorEquine Serum (ThermoFisher Scientific, Waltham, Mass.). For growth inculture, Bacto Tryptic Soy Broth (TSB) (Becton Dickinson) was used withshaking at 250 rpm. Escherichia coli strains were grown in Difco LuriaBertani Miller Broth or on LB Agar plates (Becton Dickinson).Antibiotics were added as needed at the following concentrations: N.meningitidis, erythromycin (3 microgram ml⁻¹), kanamycin (50 microgramml⁻¹), chloramphenicol (5 microgram ml⁻¹); E. coli, erythromycin (300microgram ml⁻¹), kanamycin (50 microgram ml⁻¹), chloramphenicol (50microgram ml⁻¹), ampicillin (100 microgram ml⁻¹).

Construction of OMV antigens and rabbit immunizations: Primer pairporBNdeIF/porBBamHIR (Table 3) was used to amplify the porB gene of WTstrains MC58, Cu385, BB1350, and Ch501; gene products were digested withNdeIF and BamHI and ligated into pUC18. Plasmids were verified bysequencing and transformed into MC58 porA::kan (generous gift of D. M.Granoff). Transformants were selected and PorB expression confirmed viadot blot with serotype 15 and 4 mAbs, Z15 (8B5-5-G9) and Z4 (5DC4C8G8)(National Institute for Biological Standards and Control (NIBSC), SouthMimms, Hertfordshire, England).

During confirmatory meningococcal strain sequencing, it was discoveredthat transformation with pUC18-Cu385 had resulted in a crossover eventin one of the isolates between the native MC58 gene and the Cu385 porBat the end of L1 (FIG. 4 ). This strain, designated MC58 porA::kan-OCh(also known as OCh), and the isogenic strains expressing the WT porBgenes were incubated in TSB broth and detoxified OMVs were obtained aspreviously described by incubation in 5 percent deoxycholate (Bash etal. (2000) FEMS Immunol Med Microbiol 29: 169-176). Protein content andendotoxin levels were estimated using the Pierce BCA Protein Assay Kit(ThermoFisher) and LAL Chromogenic Endotoxin Quantitation Kit(ThermoFisher), respectively, according to the manufacturer's protocols.

Rabbits were immunized with an equivalent mixture of 25 microgramsOMVs/Imject Alum Adjuvant (ThermoFisher) via intramuscular injection ofthe hind limb. Two additional immunizations were administered atthree-week intervals, and blood samples were collected one week prior tothe first immunization and two weeks following the second and thirdimmunization.

Generation of OMP deletion mutants: Primer pairsporBUPBamHIF/porBUPXbaIR, porAUPBamHIF/porAUPXbaIR, andrmpMUPBamHIF/rmpMUPXbaIR were used to PCR amplify the promoter region ofporB, porA, and rmpM, respectively. Each product was digested with BamHIand XbaI and inserted into pGEM-3Z (PROMEGA™, Madison, Wis.). Thedownstream region of each gene was then amplified usingporBDOWNSphIF/porBDOWNHindIIIR, porADOWNSphIF/porADOWNHindIIIR, andrmpMDOWNSphIF/rmpMDOWNHindIIIR; products were digested with SphI andHindIII and inserted into the gene-matched vector. The ermB, aph3A, andcat cassettes encoding resistance to erythromycin, kanamycin, andchloramphenicol, respectively, were amplified using eryXbaIF/erySphIR,kanXbaIF/kanSphIR, and chlXbaIF/chlSphIR. Digestion with XbaI/SphI andinsertion into vectors resulted in formation of the plasmids pKAM53,pKAM60, and pKAM106. Plasmids were linearized and used to transform MenBstrain MC58. Colonies were screened for double homologous recombinationvia growth on antibiotics and PCR. Deletion of PorB and PorA proteinswas confirmed via dot blot with mAbs Z15 and P7 (MN14C11.6; NIBSC). Genedeletion was confirmed in all mutants via sequencing analysis.

Construction of hybrid PorB strains: To generate chimeric porB genes(FIGS. 11A-11B), a series of overlapping PCR amplifications wasperformed. For M(1-4)C(5-8) and M(1-4)B(5-8), primer pairsporBORFXbaIF/porBF189R and porBN185F/porBORFSphIR were used to amplifyL1-L4 of MC58 porB and L5-L8 of Cu385/BB1350 porB, respectively; togenerate M(1-6)C(7-8) and M(1-6)B(7-8), primer pairsporBORFXbaIF/porBS267R and porBT263F/porBORFSphIR were used to amplifythe gene portions of the same strains. The products of each reactionwere then gel purified and a second PCR amplification was conductedusing the primer pair porBORFXbaIF/porBSphIR. The product of theoverlapping PCR was digested with XbaI and SphI and inserted intopKAM53. Proper porB insertion and ermB removal were verified byassessing colonies for ampicillin resistance and erythromycinsensitivity.

For M(1-4)C(5-6)M(7-8) and M(1-4)B(5-6)M(7-8), the same procedure wasused except that two overlapping PCR amplifications were performed. Inthe first, porBORFXbaIF/porBF189R was used to amplify MC58 L1-L4; L5-L6of Cu385 or BB1350 was amplified with porBN185F/porB267R. Hybrid L1-L6were joined together by amplification using porBORFXbaIF/porB267R. MC58L7-L8 was then obtained by PCR with porBT263F/porBORFSphIR. The finalchimeric gene product was generated by PCR amplification usingporBORFXbaIF/porBORFSphIR. Reciprocal mutant porB vectors (those bearingserotype 4 sequence in the 5′ end of the gene) for all chimeric typeswere created using the same primer pairs described above, but thestrains from which the porB loop sequences were amplified were switched.Vectors expressing the four WT PorB types and the OCh type were alsoamplified using the porBORFXbaIF/porBORFSphIR primer pair and templategenomic DNA isolated from the immunizing strains.

Following insertion of the WT and chimeric porB genes into theirrespective vectors, the aph3A cassette was PCR amplified withkanSphIF/kanSphIR and was then digested with SphI and inserted into eachplasmid. Plasmids were linearized and used to transform the OMP deletionstrain ΔB. Isolates exhibiting an erythromycin-sensitive,kanamycin-resistant phenotype indicative of a double homologousrecombination event were screened via PCR and dot blot for PorBexpression.

Recombinant PorB and PorA synthesis: Primer pair porBNdeIF2/porBBlpIRwas used to amplify each of the WT and chimeric PorB types from thepGEM-3Z-porB-kan plasmid constructs described in the section above. Eachof the products were gel purified, digested with NdeI and BlpI, andinserted into pET-28a(+) (PROMEGA™). The rPorB vectors were thentransformed into E. coli expression strain BL21(DE3) ΔompA, theconstruction of which was described previously (Qi et al. (1994) InfectImmun 62: 2432-2439). Following screening and sequencing,plasmid-bearing strains were induced for His-tagged rPorB expressionwith 0.5 mM isopropyl beta-D-1-thiogalactopyranoside (IPTG). Cultureswere centrifuged, and the pellet was suspended in 1× BUGBUSTER®ProteinExtraction Reagent (EMD Millipore, Temecula, Calif.) in Tris buffer (30mM Tris, 300 mM sodium chloride, pH 8.0), supplemented with rLysozyme(EMD Millipore). Inclusion bodies were purified via centrifugation andlysed with 8 M urea in Tris buffer. Lysates were run throughQIAGEN®Ni-NTA Superflow resin (Valencia, Calif.), and rPorB was refoldedon the column with 0.1 percent ZWITTERGENT® 3-14 detergent in Trisbuffer. Proteins were eluted with imidazole and dialyzed, and proteinconcentration was estimated by BCA. Recombinant PorA (serosubtype P1.7)was also produced from WT MC58 using the same method. porA amplificationwas achieved with primer pair porANdeIF/porABlpIR.

OMV purification: The OMP deletion mutant strains and those expressingthe chimeric (hybrid) PorB types were grown in TSB for 6 h with shaking(130 rpm), at which time cells were used to inoculate a large batch TSBculture grown for 16 h with shaking. Bacteria were heat killed for 1 hat 65 degrees Celsius, centrifuged for 30 mM at 50,000×g, and suspendedin distilled water. The cells were then broken open via French press aspreviously described (Marzoa et al. (2009) Proteomics 9: 648-656). Largedebris was removed with centrifugation at 10,000×g for 15 min, and thesupernatant was ultracentrifuged at 40,000×g for 30 min to purify OMVs.OMVs were suspended at a concentration of 1 mg/ml in distilled water andstored at −80 degrees Celsius until further use. For animalimmunizations, LOS-detoxified OMVs were obtained as previously described(Bash et al., 2000).

Immunoblots/dot blots: 500 ng of WT and chimeric (hybrid) rPorB werefractionated via SDS-PAGE and transferred to IBLOT™nitrocellulosemembranes (ThermoFisher). Alternatively, 20 micrograms of OMVs from theOMP deletion mutants were fractionated by blue native gelelectrophoresis (BNGE) (Marzoa et al. (2009) Proteomics 9: 648-656), andOMP complexes were transferred to IBLOT™ polyvinylidene difluoride(PVDF) membranes (ThermoFisher). For dot blots, OMP deletion strainswere grown on BHI plates overnight and suspended to an optical densityof OD_(600 nm)=1.0 in PBS. Following incubation for 1 h at 65 degreesCelsius to heat kill the bacteria, lysates were spotted in 5 microlitervolumes onto nitrocellulose membranes. All membranes were blocked with10 percent (%) skim milk and probed with mAbs or polyclonal antibodysera. Membranes were washed, incubated with appropriate secondaryantibody (horseradish peroxidase (HRP)-conjugated goat anti-mouse orgoat anti-rabbit IgG, Bio-Rad Laboratories, Hercules, Calif.), anddeveloped with Pierce ECL Western Blotting Substrate (ThermoFisher).

Competitive ELISAs: For competitive ELISAs, Immulon 4 HBX plates(ThermoFisher) were coated with 500 ng of rPorB or rPorA in carbonatebuffer (15 mM sodium carbonate, 35 mM sodium bicarbonate, pH 9.6)overnight at 4 degrees Celsius. Plates were washed in PBS TWEEN® (0.05percent), blocked in one percent bovine serum albumin (BSA), and probedfor binding with a mixture of either the Z15 PorB-specific or the P7PorA-specific mAbs pre-incubated (overnight at 4 degrees Celsius) withincreasing concentrations of OMVs (0-50 μg) Following overnightincubation at 4 degrees Celsius, plates were washed and incubated withHRP-conjugated goat anti-mouse antibody at 37 degrees Celsius for 2 h.Plates were developed with o-phenylenediamine dihydrochloride (SigmaAldrich, St. Louis, Mo.) and read at 490 nm. Percent inhibition wasdetermined by the calculation: ((optical density of sample—opticaldensity of control)/optical density of control)*100, wherecontrol=binding of antibody to rPorB in the absence of OMVs.

Viability tests: OMP deletion strains were streaked from overnightgrowth on plates and suspended in TSB broth at a density of OD₆₀₀=0.1.Bacteria were grown with shaking (250 rpm) and were assessed every hourfor OD₆₀₀ value for a period of 8 h. At t=0, 3, and 8 h, timescorresponding with lag phase, log phase, and early stationary phase,respectively, aliquots were obtained and serially diluted to enumerateCFUs; samples were also obtained at 8 h to obtain mRNA (see below).Representative images of CFUs at t=8 h were obtained on an Olympus SZX16stereomicroscope.

For examination of porA and porB transcripts by qPCR, mRNA was extractedfrom aliquots of the WT, ΔA, and ΔB bacterial strains using the TRIzolMax Bacterial RNA Isolation Kit (ThermoFisher) according to themanufacturer's protocol. The High Capacity cDNA Reverse TranscriptaseKit (ThermoFisher) was used to synthesize cDNA, and 50 ng of each samplewas assayed using SYBR Green PCR MasterMix (ThermoFisher). Transcriptlevels were normalized to 16S rRNA (primers shown in the Table 3), andfold change was determined relative to WT using the ΔΔCt method (Livakand Schmittgen, 2001).

Serum bactericidal assay: The exogenous complement SBA assay wasperformed in 96-well plates as previously described (Bash et al. (2014)Clin Vaccine Immunol 21: 755-761) with slight modifications. Briefly,OMP deletion strains were incubated at 37 degrees Celsius with shaking(65 rpm) in the presence of 2-fold dilutions of Z15 mAb (in 0.5 percentBSA in Hank's Buffered Saline Solution) and pooled human sera that hadbeen pre-screened for lack of endogenous killing activity. After 1 h, anoverlay of TSB with 0.7 percent noble agar was added, and plates wereincubated overnight. The following morning, CFUs were enumerated, andthe SBA titer was recorded as the reciprocal of the highest dilutionresulting in ≥50 percent killing relative to the average of the negativecontrols (bacteria that were incubated with heat inactivated sera in theabsence of Z15).

Molecular dynamics simulations: The PorB trimer structure of wild typestrain MC58 was taken from the crystal structure (PDB:3WI4), and a PorBtrimer model for strain Cu385 was built by mutating the side chains ofthe MC58 PorB structure to the Cu385 sequence. Each protein was thenembedded in asymmetric bilayers of L3 immunotype LOS in the outerleaflet and phospholipids in the inner leaflet, which mimics the N.meningitidis outer membrane. Building, assembly, and initialCHARMM-based equilibrations of these systems (MC58 and Cu385) wereachieved by CHARMM-GUI based step-by-step protocol (Jo et al. (2008). JComput Chem 29: 1859-1865, Wu et al. (2013) Biophys J 105: 1444-1455),Wu et al. (2014a) CHARMM-GUI J Comput Chem 35: 1997-2004, Wu et al.(2014b) Biophys J 106: 2493-2502., Patel et al. (2016) Biophysical J110: 930-938). 450-ns NPT (constant particle number, pressure, andtemperature) production runs were performed for both systems using NAMD(Phillips et al. (2005) J Comput Chem 26: 1781-1802) with a temperatureof 310.15 K and a pressure of 1 bar. A 2-fs time-step together with theSHAKE algorithm (Ryckaert et al. (1977) J Comput Phys 23: 327-341) wasused, and the van der Waals interactions were smoothly switched off at10-12 Angstrom (Å) by a force-switching function (Steinbach and Brooks(1994) J Comput Chem 15: 667-683), while the long-range electrostaticinteractions were calculated using the particle-mesh Ewald method(Essmann et al. (1995) J Chem Phys 103: 8577-8593). In NAMD productionrun, Langevin dynamics was used to maintain constant temperature with aLangevin coupling coefficient of 1 ps⁻¹, and a Nosé-Hoover Langevinpiston (Feller et al. (1995) J Chem Phys 103: 4613-4621, Martyna et al.(1994) J Chem Phys 101: 4177-418) was used to maintain constant pressurewith a piston period of 50 fs and a piston decay time of 25 fs. All thesimulations were performed using C36 force field for lipids (Klauda etal. (2010) J Phys Chem B 114: 7830-7843), carbohydrates (Guvench et al.(2008) J Comput Chem 29: 2543-2564, Guvench et al. (2009) J Chem TheoryComput 5: 2353-2370, Guvench et al. (2011) J Chem Theory Comput 7:3162-3180), and the TIP3P water model (Jorgensen et al. (1983) J ChemPhys 79: 926-935).

Mass spectrometry analysis: 20 micrograms of OMVs from OMP deletionstrains were fractionated by BNGE and stained with COOMASSIE® R-250.Dominant bands were cut from the gel, and in-gel digestion was performedas previously described (Jensen et al. (1999) In: Methods in MolecularBiology. A. J. Link (ed). Totowa, N.J.: Humana Press, pp. 513-53).Briefly, SDS was removed from the gel pieces with sequential washes ofacetonitrile and water, followed by drying in a speed vacuum. Sampleswere rehydrated with ammonium bicarbonate, reduced with 40 mMdithiothreitol, and alkylated with 100 mM iodoacetamide. After washing,peptides were digested overnight at room temperature with 150 ng trypsinand extracted from the gel pieces with formic acid.

Extracted peptides for each gel band were analyzed using reverse phaseone-dimensional liquid chromatography mass spectrometry (1D LC-MS/MS)with an EASY NLCTMII Proxeon nanoflow HPLC system that was coupledonline to a Q-Exactive Orbitrap mass spectrometer (ThermoFisher). Thesurvey scans were acquired in the Orbitrap analyzer at a resolution of70.000, and the fragment ions were acquired with a resolution of 17.000.MS data files were searched against the UniProtKB/Swiss-Prot N.meningitidis database (2015) supplemented with the porcine trypsinsequence using the Mascot search engine (Matrix Sciences; version2.4.0). The Mascot output files were analyzed using the softwareScaffold 4.2.0 (Proteome Software Inc.). Protein identifications wereaccepted if they could be established at greater than a 99.9 percentprobability and contained at least 2 identified peptides (Table 2).Protein probabilities were assigned by the Protein Prophet algorithm(Nesvizhskii et al. (2003) Anal Chem 75: 4646-4658), and those thatcontained similar peptides and could not be differentiated based on anMS/MS analysis alone were grouped to satisfy the principles ofparsimony.

Statistical analysis: Significance of all data was determined withtwo-way ANOVA, followed by Tukey's multiple comparisons post-test.Analysis was performed using GraphPad Prism 6 software (La Jolla,Calif.). *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001.

Example 9 Results in Animal Model Demonstrating Immune Response toNeisseria meningitidis

Deletion of the outer membrane proteins (OMPs) PorA and RmpM results indiminished PorB-specific antibody binding to the PorB molecule, aneffect that correlates with the decreased capacity of PorB-specificantibody to induce bacteriolysis in antibody-dependentcomplement-mediated killing assays. Deletion of dominant OMPs couldimpact immunogenicity and/or adjuvanticity of the meningococcal membranesurface. This effect was directly evaluated in an animal study.

Outer membrane vesicles (OMVs) were isolated from the wild type (WT)parental MC58 strain and PorA deletion mutant strains (ΔA, ΔAB, ΔAR,ΔABR, OCh) (Table 4). Two rabbits per group were immunized at threefour-week intervals with 25 μg OMVs/aluminum hydroxide (FIG. 5A).Rabbits were also immunized with equivalent concentrations of themeningococcal BEXSERO® vaccine and phosphate buffered saline(PBS)/aluminum hydroxide alone as positive and negative controls,respectively. Following termination of the study, sera were obtainedfrom each rabbit and assessed for the presence of antibodies capable ofbinding to and killing bacteria in a series of ELISAs, immunoblots, andserum bactericidal assays (SBAs).

TABLE 4 Description of OMV antigens. Antigen Name Antigen Description WTOMVs from parental MC58 PorA + PorB + RmpM + strain ΔA OMVs from WTstrain deleted for Por A expression ΔAB OMVs from WT strain deleted forPor A and PorB expression ΔAR OMVs from WT strain deleted for Por A andRmpM expression ΔABR OMVs from WT strain deleted for Por A, PorB, andRmpM expression OCh OMVs from WT strain deleted for Por A expression andcontaining a genetically mutated PorB loop 4 - loop 8 amino acidsequence relative to MC58 BEXSERO ® Vaccine from GSK Pharmaceuticalscontaining OMVs from strain NZ98/254 exhibiting different PorA and PorBamino acid sequence types relative to MC58 PBS Negative controlcontaining equimolar amounts of PBS and aluminum hydroxide given toexperimental animals

Whole cell ELISAs demonstrated the ability of all OMV antigens exceptthe negative PBS control to induce antibody responses specific to theparental MC58 strain (FIG. 5B) and four heterogeneous serogroup Bstrains (FIGS. 5C and 5D). Antisera obtained from animals immunized withΔAB or OCh OMV antigens, exhibited concentrations of antibodies bindingto whole cells of N. meningitidis that were high but diminished relativeto the other OMV antigens (FIGS. 5B-5D). Despite similarities in totalantibody binding to whole cells, the sera exhibited differingbactericidal activity. Both the B1 and B5 sera obtained from rabbitsimmunized with WT OMVs were able to mediate killing of the homologousMC58 strain and the heterologous Cu385 strain in human serumbactericidal assays (hSBAs) (FIG. 6 ). B1 antibodies were alsobactericidal against strain BB1350. A single deletion of PorA resultedin enhanced heterologous killing activity, as B2 serum antibodies wereable to kill all strains tested and B6 serum antibodies were able tokill all but one (Cu385) (FIG. 6 ). Antibodies from both of the seraobtained from animals immunized with either ΔAB or ΔABR OMVs exhibitedthe capacity to kill all five strains tested (FIG. 6 ), indicating thatdeletion of PorA and PorB in concert enhances immunogenicity ofcross-protective OMPs.

The capacity of serum antibodies from ΔAB OMV- and ΔABR OMV-immunizedrabbits to kill heterologous meningococcal strains in hSBAs suggestedthe possibility that host immune responses were being directed againstantigens that were distinct from those induced by immunization withPorA- and PorB-sufficient OMV types. To address this possibility,immunoblots were performed, probing whole cell lysates of the fivestrains tested in the hSBAs with serum antibodies from each of the siximmunized rabbits. Antibodies from B1 and B5 sera, immunized with WTOMVs, bound dominant bands at ˜17 and ˜34 kDa (FIGS. 7A-7B). Bands ofsimilar size were also bound by antibodies present in sera from animalsimmunized with ΔA (B2, B6) and OCh (B3, B7) OMVs, as well as BEXSERO®(B15, B16) (FIGS. 7A-7B). Sera from rabbits immunized with ΔAB (B9, B10)and ΔABR (B13, B14) OMVs lacked antibodies capable of binding the samebands, but contained antibodies that bound higher MW bands of ˜72 and˜95 kDa (FIGS. 7A-7B). The presence of antibodies capable of bindinghigh molecular weight bands correlated with the ability to killheterologous strains (≥4) in hSBAs, as demonstrated by the bandingprofiles of sera B2, B3, B6, B11, B15, and B16 (FIGS. 7A-7B).

Example 10 Gonococcal Vaccine from N. meningitidis Triple DeletionMutant lacking PorA, PorB, and Reduction Modifiable Protein (RmpM)Proteins

An in vivo gonococcal colonization study was conducted. In this study,mice (n=20 per group) were immunized at three fourteen day intervalswith 12.5 μg outer membrane vesicles (OMVs)/aluminum hydroxide derivedfrom (A) a wild type N. meningitidis MC58 strain containing PorA, PorB,and RmpM outer membrane proteins (OMPs), (B) strain ΔABR, in whichstrain MC58 has been deleted for PorA, PorB, and RmpM expression, or (C)strain OCh, in which strain MC58 has been deleted for PorA expressionand the amino acid sequence of PorB is genetically altered insurface-expressed loops 4-8. Mice were also immunized with equimolarvolumes of PBS/aluminum hydroxide or were unimmunized as negativecontrols. Following immunization, mice were intravaginally inoculatedwith N. gonorrhoeae strain F62 and colonization/antibody levelsmonitored over seven days (FIG. 8 ).

Mice immunized with MC58 OMVs began to clear gonococcal infection by 3days post-infection (d.p.i.), with 39% of animals cleared by day 7d.p.i. (FIG. 9A). These gross levels of clearance were greater thanthose of animals immunized with aluminum hydroxide (Alum) only orunimmunized (Unimm.) negative controls (17% and 19%, respectively),though the trend did not reach statistical significance when assessed by2-way ANOVA (FIG. 9A). In contrast, significantly more mice immunizedwith either OCh or ΔABR OMVs cleared gonococci than both negativecontrol groups, with only 23% of ΔABR OMV-immunized animals remainingcolonized by 7 d.p.i. (FIG. 9A). OCh OMV- and ΔABR OMV-immunized animalsalso exhibited a corresponding median density of colonization equivalentto 0 CFU/ml by 7 d.p.i. (FIG. 9B).

The enhanced gonococcal clearance observed when mice were immunized withPorA-deficient OMV antigens suggested that other OMPs induced theproduction of antibodies that contributed to host protection. Toidentify those antigens, western blots were performed, probing wholecells lysates of gonococcal strains F62, FA19, FA1090, and MS11, withpooled antisera obtained from animals following the third immunization.Antibodies from sera of animals immunized with MC58, OCh, and ΔABR OMVsall bound a ˜70 kDa protein present in all four gonococcal strainstested as well as the MC58 N. meningitidis

strain from which the OMVs were derived (FIG. 10 ). Antibodies from OChOMV- and ΔABR OMV-immunized mice also bound a ˜75 kDa protein that wasnot bound by serum antibodies from MC58 OMV-immunized mice (FIG. 10 ).Polyclonal antibodies present in the ΔABR pooled sera bound a ˜95 kDaprotein that was not bound by MC58 or OCh pooled sera (FIG. 10 ). Thesedata suggest unique antigens in OMV from deletion mutants contribute tothe protective effect of the OCh and ΔABR OMVs observed in the in vivoclearance model.

Example 11 Additional Protocols

Construction of Additional Strains: To construct a triple deletionmutant strain of N. meningitidis in which the outer membrane proteinsPorA, PorB, and RmpM are removed from the genome without replacement ofa selectable marker, a plasmid is constructed incorporating ˜1 kbhomologous upstream (5′) and downstream (3′) intergenic sequence of thegene from the strain to be deleted. Following incorporation of the 5′and 3′ sequences into a vector backbone, a counterselectable cassettederived from plasmid pJJ260 (Johnston, Gene 492: 325-328, 2012) bearinga kanamycin-resistance gene (nptII) and a levansucrase gene (sacB)expressed under the control of a tetracycline-inducible promoter (tetA)is inserted at the 5′/3′ sequence junction. This allows for screening ofclones by both a positive and negative selection mechanism; transformedstrains that incorporate the plasmid into the genome via a doublehomologous recombination event will exhibit resistance to the antibiotickanamycin but will be sensitive to the presence of sucrose, which isconverted to the toxic product levan in the presence of inducedlevansucrase. The complete plasmid is transformed into N. meningitidisand deletion of the gene of interest is confirmed by (1) growth onselective media containing kanamycin and (2) the absence of growth onselective media containing sucrose and chlortetracycline. To remove thecounterselectable cassette, the resulting deletion strain is transformedwith the plasmid bearing the 5′/3′ homologous sequence alone (withoutthe counterselectable cassette). Absence of the cassette is confirmed by(1) growth on selective media containing sucrose and chlortetracycline,(2) the absence of growth on selective media containing kanamycin, and(3) genetic sequencing. The process is repeated for the remaining twogenes until a PorA/PorB/RmpM triple deletion strain is constructed.

Construction of Strains and Compositions including Outer MembraneMicrovesicles and Heterologous Proteins: Proteins identified throughproteomics or bioinformatics screening that are suggestive ofOMV-mediated protection against Neisseria species will be either (1)up-regulated in the ΔABR strain via genetic manipulation or (2) producedrecombinantly and added as an additional antigen to the ΔABR OMVs. Thefollowing protocols are of use.

To construct a ΔABR OMV vaccine containing highly expressed proteinantigens of interest, the endogenous promoter region of thecorresponding gene is genetically deleted utilizing the same methodsdetailed for the clean triple deletion mutant and is replaced with aninducible promoter. The regions immediately upstream (5′) and downstream(3′) of the promoter are cloned into a plasmid, with a counterselectablecassette bearing the kanamycin-resistance gene nptII and thelevansucrase gene sacB inserted at the 5′/3′ junction. Thecounterselectable plasmid is transformed into strain ΔABR and thepromoter deletion mutant isolated via screening with kanamycin. A secondplasmid is constructed from the vector bearing the 5′/3′promoter-flanking regions. An isopropyl β-D-1-thiogalactopyranoside(IPTG)-inducible promoter (e.g. TS) is inserted directly upstream of the3′ region (encoding the open reading frame of the gene of interest) withtwo sequential genetic sequences encoding the lactose operator (lacO)and the ribosomal binding site sequence. Sequence encoding the lactoserepressor gene (lacI), which binds to the lactose operator to controlgene expression, is inserted directly downstream of the 5′ region in theopposite orientation to the TS promoter. The complete vector istransformed into the ΔABR strain containing the genomically-insertedcounterselectable plasmid and replacement of the plasmid with theinducible TS promoter is confirmed via selection on sucrose plates.Detoxified OMVs are obtained as per the protocol for the ΔABR strainexcept that IPTG is added to the medium to induce expression ofTS-controlled genes during growth in culture.

To obtain recombinant antigens of specific Neisseria proteins, genesencoding the protein of interest are inserted in-frame into anexpression vector containing a His-tag gene sequence (e.g. pET-28a(+))downstream of the inducible T7 promoter. The vector is transformed intoan Escherichia coli expression strain (e.g. BL21(DE3)); expression ofthe gene of interest is induced via growth in Luria Bertani medium inthe presence of IPTG. Bacteria are lysed and His-tagged protein ispurified by running the lysate over a nickel agarose column (Qiagen).Further purification is achieved by FPLC if necessary. Recombinantproteins are added to the ΔABR OMVs to produce a multi-componentvaccine.

In view of the many possible embodiments to which the principles of ourinvention may be applied, it should be recognized that illustratedembodiments are only examples of the invention and should not beconsidered a limitation on the scope of the invention. Rather, the scopeof the invention is defined by the following claims. We therefore claimas our invention all that comes within the scope and spirit of theseclaims.

We claim:
 1. A method of inducing an immune response to Neisseria (N.)meningitidis in a mammalian subject, comprising administering to themammalian subject an immunogenic composition comprising an effectiveamount of isolated PorA⁻PorB⁻RmpM⁻ N. meningitidis outer membranemicrovesicles, thereby inducing the immune response to the Neisseriameningitidis.
 2. The method of claim 1, wherein the immune response is aprotective immune response.
 3. The method of claim 1, wherein themammalian subject has a N. meningitidis infection and the immuneresponse is a therapeutic response.
 4. The method of claim 3, whereinthe subject has a Neisseria meningitidis infection, and theadministration of the immunogenic composition induces clearance of theNeisseria meningitidis.
 5. The method of claim 2, wherein the mammaliansubject is a healthy subject.
 6. The method of claim 1, wherein themammalian subject is a human and the outer membrane microvesicles arepurified.
 7. The method of claim 1, wherein the isolated PorA⁻PorB⁻RmpM⁻N. meningitidis outer membrane microvesicles are from a serogroup A, Bor C N. meningitidis.
 8. The method of claim 3, wherein the isolatedPorA⁻PorB⁻RmpM⁻ N. meningitidis outer membrane microvesicles are from aserogroup A, B or C N. meningitidis, and wherein the N. meningitidisinfection is of the same serogroup as the N. meningitidis outer membranemicrovesicles.
 9. The method of claim 3, wherein the isolatedPorA⁻PorB⁻RmpM⁻ N. meningitidis outer membrane microvesicles are from aserogroup A, B or C N. meningitidis, and wherein the N. meningitidisinfection is of a different serogroup as the N. meningitidis outermembrane microvesicles.
 10. The method of claim 1, wherein themicrovesicles are outer membrane vesicles, blebs, or a combinationthereof.
 11. The method of claim 1, wherein the immunogenic compositioncomprises an adjuvant.
 12. A method of treating or preventing an N.meningitidis infection in a mammalian subject, comprising: administeringto a mammalian subject an immunogenic composition comprising aneffective amount of isolated PorA⁻PorB⁻RmpM⁻ N. meningitidismicrovesicles and a pharmaceutically acceptable carrier, therebytreating or preventing the N. meningitidis infection in the mammaliansubject.
 13. The method of claim 12, wherein the microvesicles are outermembrane vesicles, blebs, or a combination thereof.
 14. The method ofclaim 12, wherein the N. meningitidis is serogroup A, B or C.
 15. Themethod of claim 12, wherein the immunogenic composition furthercomprises an adjuvant.
 16. The method of claim 12, wherein the methodprevents the N. meningitidis infection in the mammalian subject, andwherein the immune response is a protective immune response.
 17. Themethod of claim 12, wherein the method treats the N. meningitidisinfection in the mammalian subject, wherein the immune response is atherapeutic response.
 18. The method of claim 17, wherein the subjecthas a N. meningitidis infection, and the administration of theimmunogenic composition induces clearance of the N. meningitidis. 19.The method of claim 12, wherein the mammalian subject is a human and theouter membrane microvesicles are purified.
 20. The method of claim 12,wherein the microvesicles are outer membrane vesicles or blebs.
 21. Themethod of claim 12, wherein the isolated PorA⁻PorB⁻RmpM⁻ N. meningitidisouter membrane microvesicles are from a serogroup A, B or C N.meningitidis, and wherein the N. meningitidis infection is of the sameserogroup as the N. meningitidis outer membrane microvesicles.
 22. Themethod of claim 12, wherein the isolated PorA⁻PorB⁻RmpM⁻ N. meningitidisouter membrane microvesicles are from a serogroup A, B or C N.meningitidis, and wherein the N. meningitidis infection is of adifferent serogroup as the N. meningitidis outer membrane microvesicles.